Brain mitochondrial proteome alteration driven by creatine deficiency suggests novel therapeutic venues for creatine deficiency syndromes

NEUROSCIENCE RESEARCH ARTICLE Laura Giusti et al. / Neuroscience 409 (2019) 276–289

Brain mitochondrial proteome alteration driven by creatine deficiency suggests novel therapeutic venues for creatine deficiency syndromes Laura Giusti,ab1 Angelo Molinaro, cd1 Maria Grazia Alessandrì, e Claudia Boldrini, f Federica Ciregia, fg Serena Lacerenza, f Maurizio Ronci, h Andrea Urbani,i Giovanni Cioni,ae Maria Rosa Mazzoni, f Tommaso Pizzorusso,cd Antonio Lucacchini a1 and Laura Baroncellide* 1 a

Department of Clinical and Experimental Medicine, University of Pisa, I-56126, Pisa, Italy

b

School of Pharmacy, University of Camerino, I-62032 Camerino, Italy

c

Department of Neuroscience, Psychology, Drug Research and Child Health NEUROFARBA, University of Florence, I-50135, Florence, Italy

d

Institute of Neuroscience, National Research Council (CNR), I-56124, Pisa, Italy

e

Department of Developmental Neuroscience, IRCCS Stella Maris Foundation, I-56128 Pisa, Italy

f

Department of Pharmacy, University of Pisa, I-56126, Pisa, Italy

g

Department of Rheumatology, GIGA Research, Centre Hospitalier Universitaire (CHU) de Liège, B-4000, Liège, Belgium

h

Department of Medical, Oral and Biotechnological Sciences, University G. d'Annunzio of Chieti-Pescara, I-66100, Chieti, Italy

i

Institute of Biochemistry and Clinical Chemistry, Catholic university of the sacred heart, I-00168, Rome, Italy

Abstract—Creatine (Cr) is a small metabolite with a central role in energy metabolism and mitochondrial function. Creatine deficiency syndromes are inborn errors of Cr metabolism causing Cr depletion in all body tissues and particularly in the nervous system. Patient symptoms involve intellectual disability, language and behavioral disturbances, seizures and movement disorders suggesting that brain cells are particularly sensitive to Cr depletion. Cr deficiency was found to affect metabolic activity and structural abnormalities of mitochondrial organelles; however a detailed analysis of molecular mechanisms linking Cr deficit, energy metabolism alterations and brain dysfunction is still missing. Using a proteomic approach we evaluated the proteome changes of the brain mitochondrial fraction induced by the deletion of the Cr transporter (CrT) in developing mutant mice. We found a marked alteration of the mitochondrial proteomic landscape in the brain of CrT deficient mice, with the overexpression of many proteins involved in energy metabolism and response to oxidative stress. Moreover, our data suggest possible abnormalities of dendritic spines, synaptic function and plasticity, network excitability and neuroinflammatory response. Intriguingly, the alterations occurred in coincidence with the *Corresponding author at: Institute of Neuroscience, National Research Council (CNR), via Moruzzi 1, Pisa I-56124, Italy. Tel.: + 39 0 503 153199; fax: +39 0 503 153220. E-mail address: [email protected] (Laura Baroncelli).

gradient; KDM5A, Lysine-specific demethylase 5A; L, Length; MAPK1/ERK2, Mitogen-activated protein kinase 1/ Extracellular signal–regulated kinase 2; MW, Molecular weight; mTOR, Mammalian target of rapamycin; nano-ESI, Nano-electrospray ionization; nano-LC–MS/MS, Nanoscale liquid chromatography coupled to tandem mass spectrometry; Nrf2 or NFE2L2, Nuclear factor erythroid 2–related factor 2; OD, Optical density; PCr, phosphoCr; PCR, Polymerase Chain Reaction; PDIA4, Protein disulfide-isomerase; pI, Isoelectric point; PLK, Pyridoxal kinase; PP2AB, Serine/threonineprotein phosphatase 2A catalytic subunit beta; PPARGC1A, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPIA, Peptidyl-prolyl-cis-trans isomerase A; PRDX5, Peroxiredoxin 5; PRDX6, Peroxiredoxin 6; ps, particle size; PSM, Peptide spectrum-match; PSMG1, Proteasome assembly chaperone 1; R, Reverse primer; Rictor, Rapamycin-insensitive companion of mTOR; ROS, Reactive oxygen species; RuBP, Ruthenium II tris (bathophenantroline disulfonate) tetrasodium salt; SD, Standard deviation; SDS-PAGE, Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis; SDS, Sodium dodecyl sulfate; SEM, Standard error of the mean; SIM, Single ion monitoring mode; SOD1, Superoxide dismutase 1; TEMED, Tetramethylethylenediamine; TMCS, Trimethylchlorosilane; UQCRB, Ubiquinol-cytochrome c reductase binding protein; WB, Western blot; WT, Wild-type.

1

These authors equally contributed to this work. Abbreviations: 2DE, Two dimensional electrophoresis; ADP, Adenosine diphosphate; AGAT, Arginine glycine amidinotransferase; APS, Ammonium persulfate; ASDs, Autism spectrum disorders; ATP, Adenosine triphosphate; BSA, Bovine serum albumin; BSTFA, N,O-Bis (trimethylsilyl)trifluoroacetamide; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CID, Collision-induced dissociation; CK, Cr kinase; CNS, Central nervous system; Cr, Creatine; CrT, Creatine transporter; CTD, Creatine transporter deficiency; DDA, Data dependent acquisition; dNTP, Deoxynucleotide; DTT, Iodoacetamide, dithiothreitol; ECL, Enhanced chemiluminescence; EDTA, Ethylenediaminetetraacetic acid; EGLN, Egl nine homolog 1; ESRRG, Estrogen-related receptor gamma; F, Forward primer; FDR, False discovery rate; FMR1, Fragile mental retardation protein 1; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GC/MS, Gas Chromatography/Mass Spectrometry; GNAO, Guanine nucleotide-binding protein G(o) subunit alpha; HD UHR-TOF, HighDefinition Ultra High Resolution- Time of Flight; HRP, Horseradish peroxidase; I.D., Internal Diameter; I.S., Internal Standard; IB, Isolation buffer; IPA, Ingenuity Pathway Analysis; IPG, Immobilized pH https://doi.org/10.1016/j.neuroscience.2019.03.030 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 276

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developmental onset of neurological symptoms. Thus, cerebral mitochondrial alterations could represent an early response to Cr deficiency that could be targeted for therapeutic intervention. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: creatine, creatine deficiency, metabolism, mitochondria, proteomics, oxidative stress.

INTRODUCTION The high metabolic rate of the central nervous system makes it particularly vulnerable to mitochondrial derailment and oxidative stress (Attwell and Laughlin, 2001; Hall et al., 2012). A disruption of brain metabolism has been implicated in multiple psychiatric (Konradi et al., 2004; Prabakaran et al., 2004) and neurological disorders, including autism spectrum disorders (ASDs), Rett syndrome, and Angelman syndrome (Cornford et al., 1994; Trushina and McMurray, 2007; Wallace and Fan, 2010; Müller and Can, 2014; Rose et al., 2018), and the clinical phenotype of mitochondrial bioenergetic diseases is strikingly similar to that observed in a wide range of neurodevelopmental disorders (Niyazov et al., 2016). Preclinical evidence suggests that mitochondria might be a fruitful target for therapeutic intervention in various brain dysfunctions (e.g., Santini et al., 2015; Janc et al., 2016; Pei and Wallace, 2018; Rose et al., 2018). Interestingly, neuronal activity bidirectionally regulates mitochondrial production of reactive oxygen species (ROS), placing ROS as an activity tag for synaptic pruning (Cobley, 2018). The etiology of mitochondrial dysfunction is currently unclear in most brain disorders, but a tentative correlation between alterations in energy-related metabolites, such as phosphocreatine, and neuropsychological deficit has been suggested in autism (Minshew et al., 1993). Creatine (Cr) is a small metabolite holding a pivotal role in energy metabolism. Cr kinase (CK) catalyzes the reversible conversion of Cr and ATP to phosphoCr (PCr) and ADP, and the Cr-PCr system is a fundamental cytosolic buffer for ATP and a shuttle of high-energy phosphates between mitochondrial sites of production to cytosolic sites of utilization (Joncquel-Chevalier Curt et al., 2015). There is a strong link between Cr and mitochondrial function, and changes in PCr/Cr ratio finely regulate mitochondrial respiration and metabolic phenotype (Wallimann et al., 1992; Walsh et al., 2001; Dzeja et al., 2011; Chamberlain et al., 2017). In the last few years, the discovery of inherited disorders of Cr synthesis and cellular uptake (Stöckler et al., 1994; Cecil et al., 2001; Item et al., 2001) disclosed the importance of Cr supply for brain development and activity. These putatively rare diseases, characterized by Cr depletion in the cerebral compartment, share a common clinical picture dominated by neurological involvement with mental retardation, language disturbances, seizures and movement disorders (van de Kamp et al., 2014; Fons and Campistol, 2016). Cr deficiency results in disturbed metabolic activity, as well as structural abnormalities of mitochondrial organelles (Gori et al., 1988; O'Gorman et al., 1996; Nabuurs et al., 2013; Perna et al., 2016). However, a detailed molecular analysis of mitochondrial pathways possibly impaired by Cr lack is still missing. To fill this gap, we used an omic approach

to evaluate changes of protein expression in the mitochondrial fraction obtained from the brain of mice with the deletion of Cr transporter (CrT −/y mice). Considering that the phenotype of CrT −/y mice progressively worsen with age, we also assessed whether mitochondrial proteomic alterations could represent an early feature of Cr deficiency by analyzing P12 and P30 mice, two ages with relatively minimal phenotypic alterations (Baroncelli et al., 2016).

EXPERIMENTAL PROCEDURES Reagents Iodoacetamide, dithiothreitol (DTT), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), urea, thiourea, glycerol, sodium dodecyl sulfate (SDS), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), glycine and 30% acrylamide-N,N,N bisacrylamide were acquired from Applichem (Germany). IPGs pH 3–10 NL, IPG-buffer 3–10NL and dry stripcover fluid were purchased from GE Health Care Europe (Uppsala, Sweden). Enhanced chemiluminescence (ECL) detection system was purchased from PerkinElmer (MA, USA). Ruthenium II tris (bathophenantroline disulfonate) tetrasodium salt (RuBP) stain was purchased from Cyanagen. All other reagents were purchased from standard commercial sources and were of the highest grade available.

Animals CrT deficiency is an X-linked disorder. Only male mice were used this study. CrT −/y and CrT +/y mice on a C57BL/6J background were generated as previously described (Baroncelli et al., 2014). Animals were maintained at 22 °C under a 12h light–dark cycle (average illumination levels of 1.2 cd/m 2). Food (4RF25 GLP Certificate, Mucedola) and water were available ad libitum. The chow was not added with creatine (personal communication of the manufacturer). All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) and were approved by the Italian Ministry of Health (authorization number 259/ 2016-PR).

Detection of Slc6a8 mutation by PCR Genomic DNA was isolated from mouse tail using a suitable kit, following the protocol suggested by the manufacturer (DNeasy Blood & Tissue Kit, Qiagen, USA). DNA was amplified for CrT mutant and wild-type alleles using a standard PCR protocol with the following primers: F:AGGTTTCCT CAGGTTATAGAGA; R:CCCTAGGT GTATCTAACATCT; R1: TCGTGGTATCGTTATGCGCC. For PCR amplification,

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we used 300 ng of DNA in a 25-μl reaction volume containing 0.2 mM of each dNTP, 2 μM of F primer, 1 μM of R primer, 1 μM of R1 primer and 0.5 U/μl Red Taq DNA polymerase (Sigma-Aldrich, Italy). The PCR conditions were as follows: 94 °C for 4 min followed by 37 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s and a final extension at 72 °C for 7 min. Amplicons were separated using 2% agarose gel and visualized under UV light after staining with Green Gel Plus (Fisher Molecular Biology, Rome, Italy). Amplicon sizes: WT allele 462 bp; mutant allele 371 bp.

Biochemical analysis For Cr assay, mouse brains (n = 6 per CrT +/y group and n = 5 per CrT −/y group for P12; n = 4 for both groups for P30), immediately frozen on dry ice and stored at −80 °C until the analysis, were homogenized in 0.7-ml PBS buffer (SigmaAldrich, Italy) at 4 °C using a ultrasonic disruptor (Microson Heat System, NY, USA). After centrifugation (600 × g for 10 min at 4 °C) an aliquot of the homogenate (50 μl) was assayed for protein content and the supernatant used for Cr assay as previously described (Alessandrì et al., 2005). Briefly, 50 μl of saturated sodium hydrogen carbonate and 50 μl of a mixture containing 2-phenylbutyric acid (I.S.) in toluene (6.09 mmol/l; Sigma-Aldrich, Italy) were added to 200 μl of homogenate. After adding 1 ml of toluene and 50 μl of hexafluoro-2,4-pentanedione (Sigma-Aldrich, Italy) to form bis-trifluoromethyl-pyrimidine derivatives, the mixture was stirred overnight at 80 °C. The organic layer was centrifuged, dried under nitrogen and the residue derivatized at room temperature with 100 μl of BSTFA + TMCS (SigmaAldrich, Italy) injected into the Gas Chromatography/Mass Spectrometry (GC/MS) instrument. GC analyses were performed using an Agilent 6890N GC equipped with an HP5MS capillary column (0.25 mm × 30 m, film thickness 0.25 μm) and an Agilent mass spectrometer 5973N (Agilent Technologies, Italy). The mass spectrometer was set in EI-single ion monitoring mode (SIM). The ions with m/z of 192 for I.S. and 258 for Cr were used for calculation of the metabolites, using standard curves ranging 5–90 μmol/l. Data were processed by the G1701DA MSD ChemStation software. All the aqueous solutions were prepared using ultrapure water produced by a Millipore system. Differences were assessed with ANOVA test.

Enriched mitochondria preparations CrT +/y and CrT −/y mice whole brains, including the cerebellum (n = 4 per CrT +/y group and n = 3 per CrT −/y group for both P12 and P30), were rapidly collected and processed to obtain mitochondrial enriched fractions. Briefly, whole brain was weighted and homogenized in 10 vol (w/V) isolation buffer (IB) (250 mM sucrose, 10 mM Hepes, 1 mM EDTA, pH 7.5) using a Teflon-glass homogenizer (15 strokes). Sample was centrifuged at 1000 × g for 10 min at 4 °C and the resulting pellet, containing nuclei and unbroken cell, was further homogenized in IB and centrifuged at 1000 × g for 10 min at 4 °C. The resulting two supernatants were combined and centrifuged at 1000 × g for 8 min at 4 °C to remove any nuclei and cellular debris. Then, the supernatant was

centrifuged at 10,000 × g for 10 min at 4 °C to obtain enriched mitochondrial fraction. Mitochondria were resuspended in IB and washed twice. Finally, a volume of IB, depending on pellet size, was added to resuspend the mitochondrial fraction. Protein concentration was estimated by Biorad-DC assay using BSA as a standard. Mitochondrial fractions were stored at −80 °C until use.

Two-dimensional electrophoresis Two-dimensional electrophoresis (2DE) was performed as previously described (Ciregia et al., 2013). Briefly, 250 μg of proteins was filled up to 450 μl in rehydration solution, following the protocol of Rabilloud (Rabilloud, 2008) adjusted for mitochondrial solubilization. Immobiline Dry-Strips, 18 cm linear gradient pH 3–10, were rehydrated overnight in the sample and then transferred to the Ettan IPGphor Cup Loading Manifold for isoelectrofocusing according to the protocol previously described (Giusti et al., 2007). Then, Immobiline strips were equilibrated and the second dimension (SDSPAGE) was carried out by transferring proteins to 12% polyacrylamide gel, running overnight at 16 mA per gel at 10 °C. At the end of run, gels were stained with RuBP. ImageQuant LAS4010 (GE Health Care) was used for image acquisition. The analysis of images was performed using Same Spot (v4.1, TotalLab; Newcastle Upon Tyne, UK) software. The quality of gels was assessed by using the SpotCheck function. The spot volume ratios between CrT −/y and CrT +/y groups were calculated using the average spot normalized volume of the three biological replicates. The software included statistical analysis calculations.

2DE statistical analysis The significance of the differences of normalized volume for each spot was calculated by the software Same Spot including the statistical analysis by ANOVA test. We standardized the amount of each differentially expressed protein to the overall protein content measured in the mitochondrial fraction. The protein spots of interest were cut out from the gel and identified by nano-LC–MS/MS analysis.

Spot digestion and identification The gel pieces were digested as reported by Giusti et al. (2018). Samples were analyzed by LC–MS as previously described (Ciregia et al., 2015) Samples were analyzed on a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHRTOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Peptides were loaded on the EASY-Column C18 trapping column (2 cm L., 100 μm I.D., 5 μm ps, Thermo Fisher Scientific), and subsequently separated on an Acclaim PepMap100 C18 (25 cm L., 75 μm I.D., 5 μm ps, Thermo Fisher Scientific) nanoscale chromatographic column. The flow rate was set to 300 nl/min and a standard gradient from 3 to 35% of organic phase in 15′ was applied. Mobile phase A was 0.1% formic acid in H2O and mobile phase B was 0.1% formic acid in acetonitrile. The mass spectrometer was equipped with a nanoESI spray source and

Laura Giusti et al. / Neuroscience 409 (2019) 276–289

A

B

279

Hippocampus

Cr (nmol/mg pr)

Cr (nmol/mg pr)

in a maximum cycle time of 3 s. After acquiring one MS/MS spectrum, the precursors were actively *** *** *** *** 100 100 excluded from selection for 30 s. CrT+/y 80 80 Isolation width and collision energy for MS/MS fragmentation CrT-/y 60 60 were set according to the mass 40 40 and charge state of the precursor 20 ions (from 3 to 9 Da and from 20 21 eV to 55 eV). In-source refer0 0 P12 P30 P12 P30 ence lock mass (1221.9906 m/z) Age (postnatal days) was acquired online throughout Age (postnatal days) the runs. Raw data were proFig. 1. Cr brain levels in CrT+/y and CrT−/y animals at P12 and P30. Horizontal lines indicate average Cr level ± cessed using PEAKS Studio v7.5 SD for each experimental group; symbols represent individual measures for each animal. Cr levels have been software (Bioinformatic Solutions measured by GC/MS. At both ages tested, a decrease of Cr content was evident in the cerebral cortex (Two Inc., Waterloo, Canada) using the −/y Way ANOVA, post hoc Holm-Sidak method; p < 0.001 for both ages) and the hippocampus of CrT mice ‘correct precursor only’ option. (p < 0.001 for all comparisons at both ages). In addition, statistical analysis revealed no difference for the age The mass lists were searched factor (p = 0.120 for cortex; p = 0.599 for hippocampus). ***p < 0.001. against Uniprot/Swissprot database selecting Mus musculus taxoperated in positive ion polarity and Auto MS/MS mode (Data onomy (June 2017; 16,702 entries). Carbamidomethylation Dependent Acquisition, DDA), using N2 as collision gas for of cysteine was selected as fixed modification and oxidation CID fragmentation. Precursors in the range 350 to 2200 m/ of methionine and deamidation of asparagine and glutamine z (excluding 1220.0–1224.5 m/z) with a preferred charge were set as variable modifications. Non-specific cleavage state +2 to + 5 (excluding singly charged ions) and absolute was allowed to one end of the peptides, with a maximum of intensity above 4706 counts were selected for fragmentation two missed cleavages. 10 ppm and 0.05 Da were set as

Cortex

Fig. 2. Representative 2DE gel map of CrT −/y mitochondrial protein pattern from the brain of a P30 animal (A). Spots outlined indicate all differentially expressed proteins identified by nano-LC–MS/MS. The spot numbers are also reported in Table 1. (B) The histogram of the normalized OD density volumes (mean ± SEM) of four representative protein spots found differentially expressed between CrT−/y and CrT+/y. Enlarged images of the 2DE gel for these four proteins are also depicted. *p < 0.05, ***p < 0.001.

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Table 1. List of differentially expressed proteins identified by MS/MS spectrometry. ID: SwissProt accession number. Fold: average fold change is shown; min–max range is also reported in brackets. MW (th): theoretical molecular weight, pI (th): theoretical isoelectric point.

Spot n. ID

Description

Protein Fold (CrT

Cr deficiency in mice at 30 days of age 2110 Q06185 ATP synthase subunit e, mitochondrial

ATP5I

2110

Q9CQ69

Cytochrome b-c1 complex subunit 8

QCR8

1225

P62141

L-Lactate dehydrogenase B chain

LDHB

1616

P24472

Glutathione S-transferase A4

GSTA4

2016

P70349

Histidine triad nucleotide-binding protein 1

HINT1

1853

P18760

Cofilin-1

COF1

1627

Q60692

Proteasome subunit beta type-6

PSB6

2309

P08249

Malate dehydrogenase, mitochondrial

MDHM

2309

Q60931

2049

P10639

Voltage-dependent anion-selective channel protein VDAC3 3 Thioredoxin THIO

2049

P12787

Cytochrome c oxidase subunit 5°, mitochondrial

COX5A

1562

P17751

Triosephosphate isomerase

TPIS

1921

P99029

Peroxiredoxin-5, mitochondrial

PRDX5

1597

P49722

Proteasome subunit alpha type-2

PSA2

2264

P48771

Cytochrome c oxidase subunit 7A2, mitochondrial

CX7A2

1329

Q9CY64

Biliverdin reductase A

BIEA

1926

P08228

Superoxide dismutase [Cu-Zn]

SODC

660

Q8K2B3

SDHA

911

Q91YT0

911

Q8BWF0

911

P61922

Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial Succinate semialdheide dehydrogenase mitochondrial 4-aminibutirateamino transferase, mitochondrial

2233

P62984

Ubiquitin-60S ribosomal protein L40

1394

Q60932

1394

P50518

Voltage-dependent anion-selective channel protein VDAC1 1 V-type proton ATPase subunit E 1 VATE1

2331

P99029

Peroxiredoxin-5, mitochondrial

2062

Q9D855

Cytochrome b-c1 complex subunit 7

1899

P17742

Peptidyl-prolyl cis-trans isomerase A

2005

P51880

Fatty acid-binding protein, brain

1563

O08709

Peroxiredoxin-6

1563

P17751

Triosephosphate isomerase

1566

P48774

Glutathione S-transferase Mu 5

NDUV1 SSDH GABT RL40

−/y

/ CrT

5.8 (3.2–15.2) 5.8 (3.2–15.2) 1.4 (1.2–1.5) 2.1 (1.8–3.1) 1.9 (1.2–3.1) 2 (1.5–2.9) 2.6 (2.0–3.1) 2 (1.2–2.3) 2 (1.2–2.3) 1.8 (1.2–2.7) 1.8 (1.2–2.7) 1.7 (1.6–2.4) 2.2 (1.9–3.5) 1.9 (1.3–2.9) 2.4 (1.4–2.6) 1.8 (1.4–2.9) 2.2 (1.6–4.8) 1.4 (1.15–1.5) 1.6 (1.2–2.3) 1.6 (1.2–2.3) 1.6 (1.2–2.3) 1.9 (1.2–3.4) 2.1 (1.8–4.8)

2.1 (1.8–4.8) PRDX5 2.4 (1.5–5.9) QCR7 3 (1.6–9.5) PPIA 1.6 (1.3–2.6) FABP7 2.1 (1.2–2.8) PRDX6 1.6 (1.2–2.4) TPIS 1.6 (1.2–2.4) GSTM5 1.7 (1.2–3.2)

Peptides MW (th) kDa

pI (th)

pvalue

77

8

8

9.34

0.001

48

7

9

10.26 0.001

12

4

36

5.85

0.002

18

5

25

6.77

0.004

17

2

13

6.36

0.005

34

4

18

8.22

0.007

9

2

25

4.97

0.01

67

30

36

8.93

0.011

19

4

31

8.96

0.011

23

2

11

4.8

0.012

22

5

16

5.01

0.012

22

6

32

5.56

0.013

14

3

21

9.1

0.014

46

11

26

6.91

0.015

22

2

9

10.28 0.016

16

5

33

6.53

0.017

25

3

16

6.02

0.017

3

2

72

7.06

0.021

23

10

51

8.51

0.021

59

47

56

7.12

0.021

51

24

56

7.17

0.021

41

5

15

9.87

0.023

63

17

32

8.55

0.023

50

21

26

8.44

0.023

10

2

21

9.1

0.023

44

8

13

9.1

0.024

71

13

18

7.73

0.024

78

18

15

5.46

0.03

35

6

25

5.71

0.031

68

24

32

5.56

0.031

68

18

27

6.82

0.034

Coverage ) (%)

+/y

(continued on next page)

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Table 1 (continued)

Spot n. ID 1823

Q9CXZ1

1823

Q9DCJ5

983

P97807

983

Q9CZ13

1199

P05063

1262

P16858

1262

P52196

1888

P17742

1123

P63085

1139

P26516

1483

Q9DBJ1

1323

Q9WUP7

1323

Q9DB05

Description

Protein Fold (CrT

−/y

/ CrT

NADH dehydrogenase [ubiquinone] iron–sulfur pro- NDUS4 3 tein 4, mitoch (1.8–6.3) NADH dehydrogenase [ubiquinone] 1 alpha sub- NDUA8 3 complex subunit 8 (1.8–6.3) Fumarate hydratase, mitochondrial FUMH 1.5 (1.4–2.3) Cytochrome b-c1 complex subunit 1,mitochondrial QCR1 1.5 (1.4–2.3) Fructose-bisphosphate aldolase C ALDOC 1.7 (1.3–3.0) Glyceraldehyde-3-phosphate dehydrogenase G3P 1.9 (1.4–3.1) Thiosulfate sulfurtransferase THTR 1.9 (1.4–3.1) Peptidyl-prolyl cis-trans isomerase A PPIA 1.7 (1.2–3.6) Mitogen-activated protein kinase 1 MK01 1.5 (1.2–2.5) 26S proteasome non-ATPase regulatory subunit 7 PSMD7 1.8 (1.3–3.9) Phosphoglycerate mutase 1 PGAM1 1.6 (1.2–2.8) Ubiquitin carboxyl-terminal hydrolase isozyme L5 UCHL5 1.5 (1.3–1.85) Alpha soluble NSF attachment protein SNAA 1.5 (1.3–1.85)

Peptides MW (th) kDa

pI (th)

pvalue

20

4

19

10

0.035

25

5

20

8.76

0.035

44

21

54

9.12

0.035

30

11

53

5.81

0.035

82

29

39

6.67

0.038

56

21

36

8.44

0.042

36

10

33

7.71

0.042

55

9

18

7.73

0.044

54

17

41

6.50

0.045

37

8

36

6.29

0.047

29

6

29

6.67

0.047

13

4

37

5.24

0.048

79

27

33

5.30

0.048

Coverage ) (%)

+/y

Cr deficiency in mice at 12 days of age 1309 P18872 Guanine nucleotide-binding protein G(o) subunit GNAO alpha 1518 P62715 Serine/threonine protein phosphatase 2A PP2AB

64

36

40

5.69

0.034

61

23

35

5.21

0.026

5165

P0803

Protein-disulfide isomerase A4

32

16

72

5.16

0.005

1497

Q9JK23

Proteasome assembly chaperone 1

21

4

33

6.05

0.004

the highest error mass tolerances for precursors and fragments respectively. The results were filtered at 0.1% FDR PSMs.

Western blot analysis Proteins were separated by 1-D gel electrophoresis and then transferred onto nitrocellulose membranes (0.2 μm) as previously described (Ciregia et al., 2013). Anti-PPIA (Cell Signaling Technology Inc., MA, USA), anti-GAPDH (Cell Signaling Technology Inc., MA, USA) and anti-UQCRB (GeneTex Inc., CA, USA) antibodies were used at 1:1000 dilution, whereas anti-SOD1 (Cell Signaling Technology Inc., MA, USA) and anti-PRDX6 (Santa Cruz Biotechnologies, TX, USA) were used at 1:200 dilution. Finally, anti-PRDX5 (R&D Systems Biotechne, MN; USA) was used at concentration of 1 μg/ml. HRP-goat anti-rabbit (Stressgen, Belgium) and HRP-goat anti-mouse (Santa Cruz Biotechnologies, TX, USA) secondary antibodies were used at 1:10,000 dilution, whereas HRP-donkey anti-goat (Santa Cruz Biotechnologies, TX, USA) was used at 1:5000 dilution. Immunoblots

1.1 (1.1–1.4) 1.2 (1.1–1.4) PDIA4 2.1 (1.3–2.3) PSMG1 0.8 (0.7–0.9)

were developed using the enhanced chemiluminescence detection system (ECL). The chemiluminescent images were acquired using LAS4010. The immunoreactive specific bands were quantified using Image Quant-L software. In order to normalize the optical density of immunoreactive bands the optical density of total proteins was calculated. Therefore immediately after WB, the membranes were stained with 1 mM RuBP (Ciregia et al., 2017). Differences between two groups were assessed with a two-tailed t test.

Bioinformatic analysis Differentially expressed proteins were analyzed through the use of QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, CA, USA, www.qiagen.com/ ingenuity) to determine molecular and cellular functions, predominant canonical pathways and interaction network involved. Swiss-Prot accession numbers and official gene symbols were inserted into the software along with corresponding comparison ratios and p values. Networks, characterized by a score value, were generated based on functional

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Moreover, based on differentially expressed proteins the upstream regulators, whose activity appears to change in a significant manner according to the activation z-score value, were shown. Finally, to generate plausible causal networks, which explain observed expression changes, hidden connections in upstream regulators were also uncovered.

RESULTS CrT deletion leads to a widespread Cr reduction in young and adult mice We measured Cr brain levels in P12 and P30 animals using GC/MS. At both ages, we observed a significant reduction of Cr in the cerebral cortex and hippocampus of CrT −/y mice with respect to wild-type (WT) littermates (Fig. 1). No difference was detected in Cr levels measured in P12 and P30 CrT −/y mice.

Comparative 2DE analysis Fig. 3. Histograms of the normalized OD density volumes (mean ± SEM) of protein spots found differentially expressed between CrT−/y and CrT+/ y. *p < 0.05, **p < 0.01.

relationships among proteins obtained by known associations in the literature. Network with higher score and its associated biological functions and/or diseases in the Ingenuity Pathways Knowledge Base were considered for the analysis.

To identify potential mitochondrial biomarkers of Cr deficiency, we performed proteomic analysis in CrT +/y and CrT−/y mice at P12 and P30. Fig. 2 illustrates representative 2DE images of mitochondrial protein extracts. Overall an average of 900 spots was found within a non-linear pH range from 3 to 10. Fifty-five spots were found differentially expressed and the fold change was >2 for 14 of them. All spots differentially expressed resulted up-regulated in CrT −/y brain compared with CrT +/y samples. Thirty-three spots of interest

Fig. 4. Representative 2DE gel map of CrT−/y mitochondrial protein pattern from the brain of a P12 animal (A). Spots outlined indicate differentially expressed proteins identified by nano-LC–MS/MS. The spot numbers are also reported in Table 1. (B) The histograms of the normalized OD density volumes (mean ± SEM) for the protein spots found differentially expressed between CrT−/y and CrT+/y. Enlarged images of the 2DE gel highlighting the differentially expressed proteins are also shown. *p < 0.05, **p < 0.01.

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Fig. 5. Network analysis of differentially expressed proteins using the IPA software. The network revealed proteins interactions in the context of Free radical scavenging, small molecules biochemistry, neurological diseases, along with corresponding protein-to-protein direct (solid line) or indirect (dashed line) interactions, based on published literature information.

(fold change ≥1.4) were selected (Fig. 2, outlined spots) and identified by nano-LC-ESI-MS/MS. Identified proteins, with molecular weight (MW), isoelectric point (pI), and MS/MS parameters are listed in Table 1. Most of the up-regulated proteins are involved in energy metabolism, antioxidant response and proteasome catabolic pathways, with ATP synthase subunit e (ATP5I), Cytochrome b-c1 complex subunit 8 (QCR8), Glutathione S-transferase A4 (GSTA4), Proteasome subunit beta type-6 (PSB6), Peroxiredoxin-5 (PRDX5), Cytochrome c oxidase subunit 7A2 (CX7A2), Superoxide dismutase [Cu-Zn] (SODC), Cytochrome b-c1 complex subunit 7 (QCR7), Fatty acid-binding protein (FABP7), NADH dehydrogenase [ubiquinone] iron–sulfur protein 4 (NDUS4), and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 (NDUS8) showing a fold change > 2. Moreover, we also found increased levels of Cofilin-1 (COF1), Mitogen-activated protein kinase 1 (MK01) and Peptidyl-

prolyl cis-trans isomerase A (PPIA; Figs. 2B, 3). A representative image of enriched mitochondrial protein pattern at P12 is shown in Fig. 4. The comparison between CrT −/y and CrT +/y samples revealed only four spots differentially expressed and identified as Guanine nucleotide-binding protein G(o) subunit alpha (GNAO), Serine/threonine-protein phosphatase 2A catalytic subunit beta (PP2AB), Protein disulfide-isomerase (PDIA4) and Proteasome assembly chaperone 1 (PSMG1). Mass parameters are described in Table 1.

Pathway and network analysis Proteins differentially expressed at P30 were included in the Ingenuity Pathways Analysis (IPA) to identify molecular and cellular functions and to investigate whether these proteins work together in specific networks. The software generated a main network “Free radical scavenging, small molecules

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Table 2. List of upstream regulators predicted activated or inhibited based on z-score value.

Upstream Regulator

Molecular type

Activation state

Activation z-score

p-value

CD3 RICTOR EGLN KDM5A MAP4K4 FMR1 PD98059 ST1926 CD437 ESRRG Ins1 MTOR VEGFA APP PPARGC1A NFE2L2 HNF4A MYCN IL15 EGF ESRRA TP53 MYC

complex other group transcription regulator kinase translation regulator chemical kinase inhibitor chemical drug chemical drug ligand-dependent-nuclear-receptor other kinase growth factor other transcription regulator transcription regulator transcription regulator transcription regulator cytokine growth factor ligand-dependent-nuclear-receptor transcription regulator transcription regulator

Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated

−2.449 −3.162 −2.000 −2.449 −2.000 −2.236 −2.183 −2.449 −2.449 2.000 2.414 2.000 2.000 2.000 2.423 3.101 2.000 2.207 2.425 2.194 2.213 3.234 2.420

1.03E-03 9.93E-10 1.00E-04 8.94E-07 2.22E-04 1.98E-07 1.21E-02 3.22E-06 1.24E-05 3.06E-06 1.34E-04 5.65E-03 5.11E-03 8.42E-16 3.17E-05 6.85E-08 6.79E-06 6.73E-04 1.07E-05 8.60E-03 7.78E-05 3.27E-04 3.42E-04

biochemistry, neurological diseases” (Fig. 5) with a score value of 60. The downstream analysis identified key biological processes and biofunctions determined by differentially expressed proteins, i.e., neurological diseases, hereditary disorders, organismal injury and abnormalities, psychological disorders, skeletal muscle disorders, free radical scavenging, small molecules biochemistry and metabolic diseases. Moreover, canonical pathways analysis showed differentially expressed molecules involved in mitochondrial dysfunction, glycolysis, sirtuin signaling, oxidative phosphorylation and Nrf2-mediated oxidative stress response. All the proteins found to be up-regulated concurred in an upstream regulator analysis to predict whether transcription factors or genes could be activated or inactivated in agreement with the zscore value (z-score > ± 2 and p < 0.05). A list of activated and inhibited factors is shown in Table 2, whereas Fig. 6 shows their interactions with differentially expressed proteins. In particular, Lysine-specific demethylase 5A (KDM5A), Rictor, fragile mental retardation protein 1 (FMR1) and Egl nine homolog 1 (EGLN) resulted inhibited, whereas mTOR, Nuclear factor erythroid 2-related factor 2 (NFE2L2), Estrogen-related receptor gamma (ESRRG) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) resulted activated.

Western blot validation Differentially expressed proteins representing main nodes in the network were selected for validation of 2DE results by Western blot analysis on mitochondrial protein extracts of CrT +/y and CrT −/y samples. In particular, six proteins namely PRDX5, PRDX6, UQCRB, SOD1, PPIA and GAPDH were assayed using specific antibodies. For each tested protein, the optical density (OD) of the specific immunoreactive band

was determined and normalized with the corresponding total protein content and the resulting mean values ± SD were compared. Fig. 7 shows the bar graphs obtained for the validated proteins. In agreement with 2DE, all proteins resulted significantly over expressed in CrT −/y animals with respect to CrT +/y mice.

DISCUSSION Creatine transporter deficiency (CTD) is well known to cause brain Cr depletion and functional neurological deficits, but a clear picture of cellular processes and molecular changes coupling Cr shortage to brain malfunctioning is still missing. Here, we report a marked alteration of the mitochondrial proteomic landscape in Cr deficient animals, with a total of 33 identified overexpressed proteins in the brain of young (P30) CrT −/y mice. In contrast, mutant pups at P12 showed a significant change of expression only in four proteins. Many of the differentially regulated proteins at P30 are enzymes and other proteins involved in the energy metabolism chain, including glucose metabolism, the tricarboxylic acid cycle, oxidative phosphorylation and fatty acid oxidation. Moreover, we observed an increased expression of multiple antioxidant enzymes. Thus, a general pattern emerges of enhanced energygenerating pathways attempting to compensate for the power failure caused by Cr depletion. This forced metabolic phenotype is likely to originate an overload of potentially harmful byproducts of cellular metabolism, which in turn activates the antioxidant defense system. Considering the central role of Cr in energy regulation, it is not surprising that brain Cr deficit, in the attempt to compensate for loss of efficiency in the cellular network of ATP distribution, resonates in abnormal

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Fig. 6. Interactions of differentially expressed proteins with the predicted activated and inhibited transcription factors.

cellular metabolism, eventually resulting in a shift of the critical balance between free radical generation and antioxidant protection. Accordingly, it has been shown that the skeletal muscle of Arginine Glycine Amidinotransferase (AGAT) knockout animals exhibits a conspicuous increase of inorganic phosphate/ATP ratio and overall mitochondrial content, paralleled by a significant reduction of ATP levels (Nabuurs et al., 2013). Cellular metabolism and mitochondrial respiration increased in muscle fibers and brain of CrT −/y mice, with a higher number of mitochondria in CrT deficient cells (Perna et al., 2016). Moreover, pleomorphic and enlarged mitochondria, alterations of mitochondrial matrix and cristae appearance were observed at the electron microscope (Gori et al., 1988). Larger populations of motile mitochondria are present in cells with energy deficiency (Kuiper et al., 2008), potentially facilitating the recycling and elimination of damaged

mitochondria. In agreement with this, we found an upregulation of several proteins pertaining to the lysosomal catabolic and the ubiquitin proteasome pathways. IPA enrichment analysis unveiled further cellular processes and components, which are impaired in CrT deficiency. The expression of cofilin-1 was twofold increased in Cr deficient brain compared to wild-type animals, suggesting that energy stress condition leads to the accumulation of this protein. Cytoplasmic cofilin rods have been demonstrated to disrupt dendritic transport, cause loss of dendritic spines and corrupt synaptic function in other neurological disorders including Alzheimer disease, manic/bipolar disorders and autism (Chen and Wang, 2015; Woo et al., 2015; Shaw and Bamburg, 2017). Moreover, a possible link between cofilin, and mitochondrial dynamics and function has been proposed (Beck et al., 2012). Thus, neuronal misregulation of cofilin

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*

CrT+/y

et al., 2018). These data raise the possibility that normalization of CrT-/y ERK activity, by treatment with 100 ERK inhibitor peptides (Papale et 10 al., 2017; Pucilowska et al., 2018), * might rescue the CNS phenotype 8 recorded in CrT −/y mice. Our data also suggest that neuroinflammation is a crucial actor in 6 the progression of CTD disorder. We have previously observed that 4 Cr deficiency leads to an aberrant activation of microglial cells in ** 2 the brain (Baroncelli et al., 2016) * * and activated microglia can release proinflammatory cytokines, 0.04 such as peptidyl-prolyl cis-trans * isomerase A (PPIA, also known 0.02 as Cyclophilin A; Nigro et al., 0.00 2013). Research in animal models and humans provided compelling CrT+/y evidence supporting the critical function of PPIA in acute and CrT-/y chronic inflammatory diseases (Hoffmann and Schiene-Fischer, 2014). Interestingly, PPIA expression increases with aging (Nigro et al., 2013). Finally, upregulation of pyridoxal kinase (PLK) expression may be Fig. 7. Validation of PRDX5, PRDX6, UQCRB, SOD1, PPIA and GAPDH different expression in enriched mitochondrial samples of CrT−/y and CrT+/y mice by Western blot analysis. Histograms of the normalized optical involved in alterations of network density (OD) obtained in CrT−/y (n = 4) and CrT+/y (n = 5) samples are reported. Data are presented as mean ± excitability recurring in Cr defiSD. Representative bands are also depicted below the histograms. Statistical analysis was performed using a ciency syndromes (Leuzzi et al., non-parametric unpaired t test. *p < 0.05, **p < 0.01. 2013). Excessive pyridoxal phosphate concentration induced by increased PLK, indeed, may result might be involved in CTD pathogenesis. A possible trigger of in altered GABA levels (Ebadi and Klangkalya, 1979; Norris cofilin polymerization is the increased concentration of et al., 1985; Gospe et al., 1994) and/or the modification of damaged lipids, proteins and DNA as a result of oxidative specific residues on the GABAA receptor producing the stress (Bamburg and Bernstein, 2016). degeneration of GABAergic neurotransmission (Ishioka et The excessive generation of reactive oxygen species al., 1995). In addition, patients with inborn errors of metabo(ROS) and dysfunctional mitochondria might also be involved lism affecting vitamin B6 concentrations in the brain present in the overactivation of the mitogen-activated protein kinase 1 early-onset epilepsy (Wang and Kuo, 2007; di Salvo et al., (MAPK1/ERK2) signal transduction pathway (Zhu et al., 2017). Therefore, the modulation of PLK expression may reg2003; Ho et al., 2007). Depending on the cellular context, ulate seizure susceptibility in CTD patients. MAPK cascade plays a pivotal role in diverse biological funcDespite the absence of an apparent overlap in the neural tions including cell growth, adhesion, survival, differentiation, proteomic milieu at P12 and P30, IPA analysis indicates that synaptic plasticity and spine dynamics (Thomas and Huganir, antioxidative defense is precociously reinforced in the brain 2004; Ratto and Pizzorusso, 2006), but ERK pathway may of CrT −/y mice, suggesting early compensatory changes to bidirectionally affect brain function and plasticity (Silingardi preserve the balance between ROS production and their et al., 2011). A growing number of recent studies connect detoxifying enzymes. It is worth noting that we did not find a activating mutations that alter the activity of the ERK1/2 [Cr] dose-dependency for molecular changes detected in kinases to autism spectrum disorders and other syndromic the proteomic analysis, with the progressive alteration of proand nonsyndromic neurodevelopmental disorders charactertein expression not being paralleled by a decrease of Cr ized by intellectual disability (Kalkman, 2012; Borrie et al., levels in the brain tissue. Indeed, Cr levels were totally com2017), providing evidence that developmental abnormalities parable in the cortex and the hippocampus of P30 CrT −/y in neurogenesis and cortical cytoarchitecture can be mice with respect to P12 genotype-matched animals. These associated with a paradoxical increase of ERK signaling results suggest that the prolonged and continued lack of Cr (Pucilowska et al., 2015; Grissom et al., 2018; Pucilowska energy buffer might set in motion detrimental mitochondrial

OD (Normalized Volume)

200

B

R

C

X5

D

Q

U

PR

IA PP

1

D

H

D

AP

SO

G

X6

D

PR

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and molecular mechanisms leading to free radical adverse reactions that accumulate with age throughout the brain and cause the gradual shutdown of its function. These mechanisms could be involved in triggering the onset of behavioral deficits that begin to be observable at P30 in CrT −/y mice representing an early hallmark of Cr deficiency. Moreover, they could continue into adulthood contributing to the progressive deterioration of cognitive functions, accompanied by a massive lipofuscin deposition in brain cells (Baroncelli et al., 2016). This scenario, emerging from studies in the mouse, is in line with studies showing that patients with Cr deficiency syndromes have increased oxidative stress and ROS-induced apoptotic cell loss (Alcaide et al., 2011). This is the first proteomic analysis of brain samples in a CTD mouse model exploring molecular mechanisms linking Cr deficit, energy metabolism alterations and brain dysfunction. Previously, Zervou et al., 2016 used a high-throughput approach to examine Cr-dependent changes in the myocardial proteome (Zervou et al., 2016) and the metabolomics signature of CK deficiency in the heart showed a strong re-wiring of energetic phenotype (Dzeja et al., 2011). Moreover, RNA sequencing of CrT deficient fibroblasts revealed a possible impairment of extracellular matrix and cytoskeleton, suggesting a role for Cr in structural regulation of cells (Nota et al., 2014). Despite the great relevance of investigating the effects of Slc6a8 mutations at the whole body level, CrT deficiency is primarily characterized by Cr depletion in the brain and unraveling the molecular pathophysiology affecting the cerebral compartment in animal models of this disorder could be a goldmine for the discovery of novel biomarkers and druggable targets. In particular, we believe that antioxidant drugs, inflammation inhibitors, and peptides modulating cofilin activity and ERK signaling might be valid therapeutic avenues for CTD cure. Given the complexity and high dynamic range of biological samples, however, future studies will be required to confirm that cognitive and neuropsychiatric symptoms of CTD disorder are amenable to targeted drug therapy. As a final remark, we bring to attention that our findings corroborate the hypothesis that multiple disorders characterized by impaired cognition and autistic-like behavioral disturbances converge onto a few fundamental cellular processes, such as spine dynamics and the ERK/MAPK signaling cascade, affecting brain structural and functional plasticity.

AUTHOR CONTRIBUTION LG, GC, MRM, TP, AL and LB conceived the study. LG, MRM, TP, AL and LB designed the experiments. LG, AM, MGA, CG, FG, SL, MR, AU and LB carried out the research. LG, MGA, CG, FG, SL, MR, AU analyzed the data. LG, AM, MRM, TP AL and LB wrote the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

ACKNOWLEDGMENTS This research has been supported by grant GR-201602364378 funded by the Italian Ministry of Health to LB and

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University of Pisa. We thank Francesca Biondi for animal care.

COMPETING INTERESTS No competing interests to disclose.

ETHICS APPROVAL All experiments were carried out in accordance with the European Directive of 22 September 2010 (2010/63/UE) and were approved by the Italian Ministry of Health (authorization number 507/2018-PR).

DATA AVAILABILITY The datasets generated during the current study are available from the corresponding author on reasonable request.

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(Received 1 February 2019, Accepted 12 March 2019) (Available online 25 April 2019)