neurotoxin-induced neuropeptide perturbations in striatum of neonatal rats

Neurotoxin-Induced Neuropeptide Perturbations in Striatumof Neonatal RatsOskar Karlsson,*,†,‡,# Kim Kultima,#,†,§ Henrik Wadensten,† Anna Nilsson,† Erika Roman,†

Per E. Andren,† and Eva B. Brittebo†

†Department of Pharmaceutical Biosciences, Uppsala University, SE-751 24 Uppsala, Sweden‡Department of Environmental Toxicology, Uppsala University, SE-752 36 Uppsala, Sweden§Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University,SE-751 85 Uppsala, Sweden

*S Supporting Information

ABSTRACT: The cyanobacterial toxin β-N-methylamino-L-alanine (BMAA) is suggested to play a role in neuro-degenerative disease. We have previously shown that althoughthe selective uptake of BMAA in the rodent neonatal striatumdoes not cause neuronal cell death, exposure during theneonatal development leads to cognitive impairments in adultrats. The aim of the present study was to characterize thechanges in the striatal neuropeptide systems of male and femalerat pups treated neonatally (postnatal days 9−10) with BMAA(40−460 mg/kg). The label-free quantification of the relativelevels of endogenous neuropeptides using mass spectrometryrevealed that 25 peptides from 13 neuropeptide precursors weresignificantly changed in the rat neonatal striatum. The exposureto noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, andchanges in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derivedpeptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level ofpeptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, infemale pups. Because several of these peptides play a critical role in the development and survival of neurons, the observedneuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptidechanges suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggeststhat neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on thedeveloping brain.

KEYWORDS: neurotoxin, BMAA, neonatal striatum, neuropeptide, ALS/PDC, cyanobacteria, sex differences, cortistatin

■ INTRODUCTION

Cyanobacteria exist as free-living or symbiotic organisms interrestrial and aquatic environments worldwide. These organ-isms produce several cyanotoxins and are capable of massiveproliferation, with large cyanobacterial blooms in various types ofwaters. Most cyanobacteria produce the neurotoxic nonproteinamino acid β-N-methylamino-L-alanine (BMAA).1,2 BMAA hasbeen detected in several water systems, including temperateaquatic ecosystems, and in mollusks and fish used for humanconsumption, suggesting that BMAA bioaccumulates in aquaticfood chains.3,4 Exposure to BMAA has been implicated in theetiology of Amyotrophic lateral sclerosis/Parkinsonism-demen-tia complex (ALS/PDC) on the island of Guam5,6 and in ALSand Alzheimer’s disease in North America.7

BMAA is an ionotropic and metabotropic glutamate receptoragonist that induces neuronal degeneration via excitotoxicmechanisms, although other mechanisms of toxicity might also

be involved, such as oxidative stress or the misincorporation ofthe beta-amino acid into protein.8−11 At low concentrations,BMAA is not considered to be acutely neurotoxic to adultrodents12,13 and the access of BMAA to the adult rodent brain isreported to be limited.14,15 In contrast, autoradiographic studiesrevealed that 3H-BMAA-derived radioactivity is transferredacross the blood−brain barrier in neonatal mice, with a distinctlocalization in specific brain regions, such as the striatum and thehippocampus.16 BMAA treatment of neonatal rats duringdevelopment induced transient behavioral changes, such asdisturbed motor function and hyperactivity in neonates16 andlong-term cognitive impairments and changes in neuronalprotein expression in adults.17 A high dose (460 mg/kg) ofBMAA induced acute neuronal cell death in the neonatal

Received: October 30, 2012Published: February 15, 2013

Article

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© 2013 American Chemical Society 1678 dx.doi.org/10.1021/pr3010265 | J. Proteome Res. 2013, 12, 1678−1690

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hippocampus, retrosplenial and cingulate cortices, but no neuronalcell death was observed in the neonatal striatum.18 The striatumis important for several cognitive processes19,20 and even subtleeffects during the development of the brain region mightcontribute to the observed cognitive impairments in learning andmemory in adult animals following neonatal exposure toBMAA.18,21

To elucidate the mechanisms of BMAA-induced changesfollowing neonatal exposure, more detailed studies on the earlyeffects of BMAA in the developing brain are needed. The highand selective uptake of 3H-BMAA in the striatum, which isimportant for motor function and cognitive tasks,19,22 makes thisbrain region an interesting candidate for studies of BMAA-induced effects during development. The aim of the presentstudy is to examine the effects of BMAA on neuropeptide levelsin the neonatal striatum using a label-free mass spectrometry-based approach. The results showed that neonatal exposure toBMAA induced a dose-dependent increase of VGF-derivedpeptides associated with important developmental changes in thebrain. We also showed that neonatal exposure to BMAAincreased the relative levels of the protachykinin 1-derivedC-terminal-flanking peptide, substance P and neurokinin A infemale pups. Because several of these peptides play a critical rolein the differentiation and survival of neurons, the observedneuropeptide changes in the neonatal striatum might mediateBMAA-induced behavioral effects.16,18,21

■ MATERIALS AND METHODS

Chemicals

Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO). β-N-Methylamino-L-alanine (L-BMAA) hydrochloride (≥97%) was used.Animal Experiments

Pregnant outbred Wistar rats were obtained from Taconic (Ejby,Denmark). Each dam was housed alone in a standard cage (59 ×38× 20 cm) containing wood-chip bedding and nesting material.The animals were maintained on standard pellet food (R36Labfor; Lantmannen, Kimstad, Sweden) and water ad libitum,and housed in a temperature- and humidity-controlled environ-ment with a 12-h light/dark cycle (lights on at 6 a.m.).After the day of birth (postnatal day (PND) 0), all male and

female pups in each litter were randomly assigned to the controlor to one of the BMAA treatment groups. The male pups weregiven one daily subcutaneous injection (20 μL/g) of BMAA at40mg/kg (M40; corresponding to 50mg/kg BMAAHCl; n = 8),150 mg/kg (M150; corresponding to 200 mg/kg BMAA HCl;n = 8), or 460 mg/kg (M460; corresponding to 600 mg/kgBMAA HCl; n = 8) freshly dissolved in Hanks’ balanced saltsolution, or vehicle (MC; n = 8), for 2 days (PNDs 9 and 10).The female pups were treated with 150 mg/kg BMAA (F150;n = 8), or vehicle (FC; n = 8), for 2 days (PNDs 9 and 10). Therat pups were sacrificed by decapitation at 24 h after the lastBMAA treatment. The brains were dissected on ice and slicedmanually in a cooled rat brain matrix (coronal sections, 1 mmslots; ASI Instruments, Inc., Warren, MI) using razor blades. Thecaudate putamen (striatum) was dissected from the sections withthe guidance of a neonatal rat brain atlas.23 Striatum wascollected from sections between approximately 6.2 and 5.3 mm,but not beyond the anterior commissure. The tissues wereimmediately frozen on dry ice and stored at −80 °C until furtheruse for peptide analysis.

The Uppsala animal ethical committee approved all animalexperiments, which were performed in accordance with theguidelines of the Swedish legislation on animal experimentation(Animal Welfare Act SFS1998:56) and European Unionlegislation (Convention ETS123 and Directive 86/609/EEC).

Tissue Extraction and Sample Preparation

The frozen brain samples were denatured at 95 °C for 30−40 susing a rapid heat transfer inactivation instrument (Stabilizor,Denator AB, Gothenburg, Sweden) to completely preventprotein degradation.24 The tissue samples were subsequentlytransferred to low-retention Eppendorf tubes, suspended inprechilled extraction solution (7.5 μL 0.25% (v/v) aqueousacetic acid/mg tissue) and homogenized by sonication (Vibracell 750, Sonics & Materials, Inc., Newtown, CT) in an ice-bathfor 30 s.25 Each sample suspension was centrifuged at 14 000g for40 min at 4 °C to remove insoluble material. The supernatantwas transferred to Microcon 10 kDa cutoff spin columns (YM-10,Millipore, Bedford, MA) and centrifuged at 14 000g for 90 min at4 °C. The resulting peptide filtrate was frozen at −80 °C untilfurther analysis.

Randomized Block Design of Mass Spectrometry (MS)Analysis

For quantitativeMS analyses, the samples were run in a restrictedrandomized block design. Thus, one biological sample from eachtreatment group (six groups) was randomly selected andconsecutively analyzed (as one block of samples). Thisprocedure was repeated until all samples were analyzed. Theorder in which the samples within each block were analyzed wasrestricted (i.e., the order in which the samples from differenttreatment groups were injected was different in all blocks).A restricted randomized block design was used to avoidsystematic bias due to flow changes in the LC system andminimize the effect of a potential carryover effects betweensamples affecting the relative peptide calculations. Prior to eachblock of samples, a blank run (0.25% (v/v) aqueous acetic acid)was performed and a reference sample of pooled material wasanalyzed as every seventh sample for internal quality control.After all forty-eight biological samples were analyzed, theprocedure was repeated, constructing new blocks of samplesand analyzing all samples a second time. This procedure wasundertaken to minimize the number of missing values in eachsample and to facilitate the estimation of both technical andbiological variations. All samples were analyzed without changingthe analytical column.

Liquid Chromatography (LC)

An aliquot (5 μL) of the peptide filtrate was obtained from eachanimal and analyzed on a nano-LC system (Ettan MDLC, GEHealthcare, Uppsala) using a nano-electrospray ionization(nano-ESI) interface coupled with an Q-Tof-2 (Waters,Manchester, U.K.) or a linear ion trap (LTQ; Thermo Electron,San Jose, CA) mass spectrometer for quantification andidentification, respectively. The sample was injected and desaltedon a precolumn (300 μm inner diameter (i.d.) × 5 mm, C18PepMapT, 5 μm, 100 Å, LC Packings, Amsterdam, TheNetherlands) at a flow rate of 10 μL/min for 10 min. A 15-cmfused silica emitter with a 75 μm i.d. and a 375 μmouter diameter(New Objective; Woburn, MA) was used as the analyticalcolumn. The analytical columnwas packed in-house with reverse-phase Reprosil-Pur C18-AQ 3-μm, 120 Å, resin (Dr. Maisch,GmbH; Ammerbuch-Entringen, Germany) using a pressurizedpacking device (ProxeonBiosystems;Odense, Denmark). Buffer A

Journal of Proteome Research Article

dx.doi.org/10.1021/pr3010265 | J. Proteome Res. 2013, 12, 1678−16901679

(0.25% (v/v) aqueous acetic acid) and Buffer B (84% acetonitrileand 16% of 0.25% (v/v) aqueous acetic acid) were used as mobilephases. For the separation and elution of the peptides, a 40-mingradient from 3% to 65% Buffer B at a flow rate of approximately200 nL/min was applied.

Peptide Quantification

The quantitative analysis of the peptide samples was performedon the Q-Tof-2 instrument. The label-free quantification ofrelative peptide levels was conducted as described previously8−11

with minor modifications. Brifely, the mass spectrometer wascalibrated according to the manufacturer’s recommendationsusing a PEG solution (Fluka, Switzerland). MS data werecollected in a continuous mode in the m/z range 300−1000 for45 min. The raw MS data were converted into ASCII files usingthe Data Bridge module available in the MassLynx software(V3.5, Micromass) and imported into DeCyder MS2.0 (GEHealthcare). Ion peaks were automatically detected (parameters:elution time 0.2 min, typical peak resolution 8000, acceptedcharge states 1−12, signal-to-noise cutoff of 4, backgroundsubtracted quantification (smooth surface). Thereafter, theDeCyder MS 2.0 time alignment function was applied, withthe following parameters: max stretch/compress, 1 min; maxleader, 10%; stretch/compress penalty, 0.1. The ion peaks of thetime aligned intensity maps were subsequently matched with thefollowing tolerances: time ±1 min and m/z ± 0.2 Da. Prior todata export, the identified peptides were manually controlled andthe mismatched peaks were removed from subsequent dataanalyses.

Peptide Identification

Nano-LC LTQ MS/MS analysis was performed on each samplefor the identification of peptides. The same nano-LC system andsetup as used for the peptide quantification was applied. TandemMS data were obtained in a data-dependent manner, withcontinuous switching between full MS (m/z 300−2000), zoom(most intense peak in full scan) and MS/MS scans, where themost intense peak is selected twice in a time window of 40 sbefore inclusion on an exclusion list during a 150-s period. Thetandem mass spectra were converted into data files usingXcalibur (Version 2.0 SR2) and further compiled into mgf filesusing an in-house developed script. The obtained MS/MSspectra were searched against the following sequence collectionsfrom the SwePep database,26 SwePep precursors, SwePeppeptides and SwePep prediction27 or against the UniProtKnowledgebase (UniProtKB) using X! Tandem28 for the specificidentification of endogenous peptides. The following settingswere used for the database search: peptide mass tolerance of +2/−0.5 Da; fragment mass tolerance of ±0.5 Da; unspecificcleavage (SwePep precursor), respectively, no-cleavage (SwePepprediction); possible post-translational modifications (N-termi-nal acetylation, N-terminal pyro-glutamic acid derived fromglutamine, C-terminal amidation, oxidation of methionine andtryptophan, phosphorylation of serine, tyrosine and threonine).A significance threshold of log <−2 was used for individualpeptides. The peptides included here, which did not reach thisthreshold, have beenmanually verified and anMS/MS analysis ofthese peptides is shown in the Supporting Information.In an effort to identify as many neuropeptides as possible, two

of the samples (No 12MC andNo 49M460) were analyzed for asecond time on an Orbital Trap Mass Spectrometer (LTQ XL;Thermo Scientific, San Jose, CA). The samples were analyzed ona nano-LC system using nano-ESI (Surveyor MS Pump Plus;Thermo Scientific, San Jose, CA). As mobile phases, Buffer A

(2% (v/v) acetonitrile in 0.1% (v/v) aqueous formic acid) andBuffer B (0.1% (v/v) formic acid in acetonitrile) were used. Thesamples were injected and desalted on a precolumn. Theanalytical column was a Biopshere C18 120A, 360/75-μmcolumn, with 15-cm length. For the separation and elution of thepeptides, a 90-min gradient from 8 to 40% Buffer B at a flow rateof approximately 200 nL/min was applied. Tandem MS datawere acquired in a data-dependent manner, with continuousswitching between full MS (m/z 300−2000) and zoom scans,followed by 5 MS/MS scans, where the most intense peak isselected before inclusion on an exclusion list during a 60 s period.The analysis confirmed the identity of 6 peptides that werevaguely identified in the main run. The MS/MS spectra for thesepeptides are provided in the Supporting Information.

Normalization and Data Analysis

Normalization was conducted on log2-transformed data exportedfrom the DeCyderMS software. To correct for global intensitydifferences between peptide runs, the data were normalized intwo steps.29 Briefly, a linear regression was fitted for eachindividual run to a median run constructed of all median peakvalues for ions matched in >10% of all runs. On the basis of thelinear regression equation, new values were predicted for eachrun. A locally weighted polynomial regression (Lowess) wassubsequently fitted for each matched peptide against the runorder, and the mean value across all runs was added to retain thenative intensity dimension. For each matched peptide, aproportion of 0.2 neighbors (runs), weighted by their distanceto the measurement, were used to controll the smoothness of thefit. Two technical replicates and three biological samples wereremoved due to low peak intensity or high background, resultingin a small fraction of peaks successfully matched to all othersamples. These qualitymeasures generated the following samplesper group:MC (n = 8),M40 (n = 7),M150 (n = 8),M460 (n = 7),FC (n = 7) and F150 (n = 8). All LC−MS analyses includemissing peak values due to technical reasons. Consequently, onlythe peaks/peptides, which were identified, correctly aligned andmatched in at least five samples in each group, were subjectedto downstream data analyses, assuring that the number ofobservations in all groups is similar and excluding peaks/peptideswith a large number of missing values.To examine differences in the peak expression between the

groups three different linear models were employed. Differenttypes of models were used to obtain the best estimates for thedifferent biological questions considered.(i) In the first model, the summation of the group means for

males (four groups) was considered as fixed effects and individualsamples were defined as random effects. This model provides thebest estimate of the treatment effect of BMAA in males only.(ii) In the second model, the summation of the group means

for sex (two groups), the treatment groups in both sexes (M150and F150) and the interaction between sex and treatment wereconsidered as fixed effects. Individual samples were defined asrandom effects. This model considers the interaction effectbetween males and females and the effect of 150 mg/kg BMAA,which is the only dose that was measured in both males andfemales. In the case of a significant interaction effect, females andmales respond differently to BMAA exposure.(iii) In the thirdmodel, the summation of the groupmeans (six

groups) for all different groups was considered as fixed effectsand individual samples were defined as random effects tocompare all six groups. Based on this model the varianceestimates are presented for the biological samples and technical



replicates based on the repeated measurements of the samples(within).The marginal sum of squares was calculated to asses the main

and interaction effects. Restricted maximum likelihood estima-tion was used. An F-test was, performed to test the significance ofeach model. An F-test, followed by t testing was used forintergroup comparisons of single peptides, using control males(MC) as a baseline for all comparisons to display all comparisonsin one figure using the same baseline. Because the error bars inFigures 2−5 represent 95% confidence intervals, the groups witherror bars not overlapping the baseline were considered asstatistically significant compared with the control males. Groupswith error bars not overlapping the average estimate of othergroups were also statistically significant, replacing the need formultiple pairwise comparisons. To facilitate interpretation,relevant significant differences compared with MC and F150compared with FC are indicated with asterisks; *p < 0.05, **p <0.01, ***p < 0.001.A multivariate projection-based, principal component analysis

(PCA) was used to visualize and further evaluate the data. Thismethod was designed to extract and display the systemicvariation in a data set. The results from the PCA are presented inscore, loading and contribution plots. The score plot summarizedthe possible relationships between the individuals, and theloading and the contribution plots identified the variablesimportant for these relationships. The average of the tworeplicate measurements of each sample and peptide were used inthe analysis. Linear models ii and iii were applied to PCA scores.The scores and loadings from statistically significant t-scoreswere used to calculate the relative contribution scores. Thisapproach was applied to the data for the identified peptides (n =103). The precursor level was calculated as the average of thefold-change from all peptides derived from the same precursor.The nlme30 library available in the R31 and SIMCA-P (version 12,Umetrics AB, Umea, Sweden) software was used for thestatistical analysis. The resulting fold-change values are listedon log2 scale in the tables and figures, and to facilitateinterpretation on linear scale in the text.

■ RESULTS

The quality marker stathmin (2−20), which is typically present atpost-mortem protein degradation,24 was not detected in any ofthe samples. All samples were therefore considered devoid ofproteolytic peptides produced post mortem from high abundantproteins.

Striatal Neuropeptides in BMAA-Treated Neonates

In total, 103 peptides from 27 different precursors were identifiedand relatively quantified in this study (Supporting InformationTable). The relative levels of 25 peptides derived from 13neuropeptide precursors were significantly altered in thestriatum of rat pups treated neonatally with BMAA and/or inmales and females (Table 1). All but one peptide (tubulinbeta-2A/B chain) originated from precursors known tocontain neuropeptides.A global analysis of the 103 identified peptides using PCA

resulted in seven components, explaining 80% of the variation.On the basis of these PCA scores, we examined potential sexdifferences using the M/F150 dose (model ii). The resultsshowed a statistically significant (p = 0.05) sex/treatmentinteraction effect in component three and a significant (p < 0.05)treatment effect in component five. Applying model iii to thePCA scores revealed that components three (8.9%), five (5%)

and six (4.1%) were statistically significant. The M150 andM460treatment groups were well separated from the M40 and MCgroups using components three and five (Figure 1A).Comparing the male controls to treated males revealed that

the peptides derived from the precursors VGF, cholecystokinin,proSAAS and the chromogranins chromogranin-A and secretog-ranin-1, -2, and -3 contributed most to the observed treatment-related differences (Figure 1B,C). The peptides derived from theprecursors proenkephalin-A, thymosin beta-10, thymosin beta-4and the chromogranins contributed most to the sex/treatment-related differences (Figure 1B,D).

Dose-Dependent Increase of Peptides from theVGF-Precursor

Neonatal BMAA treatment induced a statistically significantincrease in the relative level of four peptides from the VGFprecursor in the striatum. The levels of the peptides, ASWGEFQ(novel, VGF 211−217), VPERAPLPPSVPSQFQ (novel, VGF220−235), NAPPEEPVPPPRAAPAPTHV (NERP-432), PPP-RAAPAPTHV (novel, VGF 496−507), were dose-dependentlyincreased, with a 1.91-fold increase at the highest dose comparedwith the vehicle control group (Table 1 and Figure 2). Five otherpeptides from the VGF precursor, including the known peptideVGF (180−194), were detected, and the levels of most of thesepeptides were increased following BMAA exposure, particularlyin the M460 group. In addition, the VGF precursor level wasstatistically increased in the M150/F150 and M460 groupscompared with the vehicle control group.

Increased Levels of Peptides from the CholecystokininPrecursor

Neonatal BMAA treatment induced a statistically significantincrease in the relative level of the cholecystokinin-derivedpeptide QPVVPVEAVDPMEQ (part of propeptide 21−45) inthe striatum, with a 1.95-fold increase in the M460 groupcompared with the vehicle control group. The levels of two othercholecystokinin-derived peptides, including the longer form ofthe cholecystokinin-33 peptide, were also increased (SupportingInformation Table).

Both Increased and Reduced Levels of Peptides fromChromogranin and Secretogranin Precursors

Neonatal BMAA treatment induced a statistically significantchange in the relative level of peptides from chromogranin andsecretogranin precursors in the striatum. The two peptidesderived from the chromogranin-A precursor were differ-entially changed in the M460 group. The LEGEDDPDR-SMKLSFRA (novel, CMGA 377−393) peptide was signifi-cantly reduced (0.77-fold) in the M460 group (the same trendwas observed in the M150 and M40 groups). In contrast, theLEGEDDPDRSMKLSF (CMGA 377−391) peptide, lackingtwo C-terminal amino acids, was significantly increased (1.39-fold) in the M460 group compared with the vehicle controlgroup (Table 1).Furthermore, the levels of the four peptides derived from the

secretogranin-1 precursor were differentially changed in theM460 group compared with the vehicle control group. TheHTEESGEKHNAFSN (novel, SCG1 186−199) and DEGHDP-VHESPVDTA (novel, SCG1 437−451) peptides were bothsignificantly reduced (0.71-fold and 0.74-fold, respectively),whereas both the SEES(Phos)QEKEY (novel, SCG1 371−380)and YPQSKWQEQ (novel, SCG1 454−463) peptides weresignificantly increased 1.18-fold and 1.16-fold, respectively(Table 1 and Supporting Information Figure 1).



Table1.The

Levelsof25

Neuropeptidesfrom

13NeuropeptidePrecursorsintheNeonatalStriatumat24

hafterthe

LastT

reatmentinMaleandFemaleRatsAdm

inisteredBMAA

onPND9−

10a,b

treatm

entgroups

ANOVA(p)

M40-M

CM150-MC

M460-MC

FC-M

CF150-M

CF150-FC

variance

precursorc

peptidename

sequence

N-term−C-term

cleavage

sites

male i

all iii

int ii(p)

log 2

,ip i

log 2

,ip i

log 2

,ip i

log 2

,iii

p iii

log 2

,iii

p iii

log 2

,iii

p iii

sample iiiwith

iniii

7B2

C-terminalpeptide(5−13)

202 FSE

EEKEP

E210

KKSV

PH-C

-term

0.082

0.019

0.013

−0.0250.861−0.3340.021−0.1760.224

−0.4570.002−0.2380.090

0.2190.131

0.043

0.062

CCKN

Propeptid

e21−45

(1−14)

21QPV

VPV

EAVDPM

EQ34pG

luQ1LA

-RA

0.028

0.097

0.231

0.5110.090

0.5640.054

0.9640.004

0.5120.124

0.4710.121−0.0410.903

0.219

0.179

CMGA

CMGA377-391

377 LEG

EDDPD

RSM

KLS

F391

KR-RA

0.002

0.002

0.415

−0.0060.965−0.0040.975

0.4729.21

×10

−04−0.1490.270

0.0250.851

0.1740.203

0.045

0.043

CMGA

CMGA377−

393

377 LEG

EDDPD

RSM

KLS

FRA393

KR-RA

0.012

0.075

0.433

−0.1630.131−0.1810.085−0.3770.001

−0.0760.534−0.1000.405−0.0230.851

0.044

0.023

MCH

Neuropeptide-glutam

icacid-isoleucine

(NEI)

131 EIG

DEE

NSA

KFP

I143AmideI13

KR-GRR

0.002

0.015

0.483

−0.0080.958

0.1220.400

0.5756.12

×10

−4

0.0220.906

0.3490.056

0.3270.082

0.119

0.012

MCH

MCH131-144

131 EIG

DEE

NSA

KFP

IG144

KR-RR

0.001

0.002

0.991

−0.2010.216−0.0390.800

0.4970.004

0.1030.516

0.0650.657−0.0380.804

0.067

0.022

PENK

PENK114-133(7−20)

120 VEP

EEEA

NGGEILA

133

KKMDEL

YP-K

R0.060

0.027

0.516

−0.1690.117−0.0580.567

0.1350.201

0.1710.099

0.0310.756−0.1400.175

0.032

0.013

PENK

Propeptid

e198-209(1−10)

198 SPQ

LEDEA

KE207

KR-LQ

KR

0.001

0.004

0.053

−0.2780.168−0.1900.323

0.5710.007

−0.2070.391

0.3620.124

0.5690.022

0.204

0.015

PENK

Propeptid

e219-229(1−7)

219 VGRPE

WW

225

RR-M

DYQKR

0.460

0.024

0.313

−0.1120.283

0.0330.739

0.0490.637

0.3270.010

0.1690.162−0.1580.204

0.040

0.025

SCG1

SCG1186-199

186 H

TEE

SGEK

HNAFS

N199

KK-K

R0.002

0.002

0.341

0.0270.819−0.0770.517−0.4846.16

×10

−4−0.2400.049−0.1540.184

0.0860.476

0.034

0.029

SCG1

SCG1371-380

371 SEE

SQEK

EY380Ph

osS4

PR-K

R0.035

0.032

0.063

0.1990.097−0.0630.575

0.2440.045

−0.1140.389

0.2060.114

0.3200.020

0.041

0.046

SCG1

SCG1437-451

437 D

EGHDPV

HES

PVDTA451

KRLL

-KR

0.009

0.013

0.206

0.1060.481−0.1410.335−0.4300.006

−0.2020.143−0.0790.555

0.1230.377

0.038

0.054

SCG1

SCG1454-462

454 YPQ

SKWQEQ

462

KR-EK

0.024

0.047

0.099

0.1310.200−0.0940.337

0.2090.044

−0.0900.409

0.0780.461

0.1680.131

0.030

0.025

SCG2

SCG2598-612

598 YLN

QEQ

AEQ

GREH

LA612

KVLE

-KR

0.011

0.036

0.026

−0.0510.536−0.1150.155−0.2850.002

−0.1880.056−0.0080.930

0.1800.069

0.025

0.018

SCG2

SCG2599-612

599 LNQEQ

AEQ

GREH

LA612

KVLE

Y-K

R0.009

0.015

0.003

−0.0450.622−0.2180.019−0.2960.003

−0.2750.015

0.0030.978

0.2780.015

0.020

0.045

SCG3

SCG338-53

38EL

SAER

PLNEQ

IAEA

E53

NR-A

DK

5.78

×10

−54.54

×10

−5

0.596

0.1040.402

0.2600.036

0.6728.48

×10

−6−0.0390.766

0.1160.364

0.1550.243

0.059

0.009

SMS

Somatostatin

-28(1−12)

89SA

NSN

PAMAPR

E100

QR-R

K0.029

0.068

0.295

0.0400.868−0.0200.933−0.6440.013

−0.1890.495

0.2150.425

0.4040.150

0.257

0.050

TBB2C

Tubulin

beta-2Cchain

168-185

168 SVVPS

PKVSD

TVVEP

YNA185

MNTF-T

LSV

0.009

0.029

0.089

−0.0510.632

0.0680.510−0.3080.007

0.1330.313−0.1390.280−0.2720.044

0.057

0.012

TKN1

C-terminal-flanking

peptide

111 ALN

SVAYER

SAMQNYE126

KR-RR

0.164

0.015

0.676

−0.0500.727

0.2450.085−0.0050.971

0.0890.562

0.4230.005

0.3350.034

0.075

0.009

TYB10

TYB

1030-44

30PT

KET

IEQEK

RSE

IS44

KNTL-C

-term

0.235

0.028

0.054

0.0050.972−0.2280.076−0.0690.592

−0.4180.004−0.2320.089

0.1870.181

0.058

0.023

TYB4

TYB

428-44

28PL

PSKET

IEQEK

QAGES

44KN-C

-term

0.468

0.027

0.009

−0.0350.795−0.1930.142−0.1000.455

−0.5370.005

0.0710.684

0.6080.002

0.103

0.033

VGF

VGF211-217

211 ASW

GEF

Q217

WR-A

R1.56

×10

−51.61

×10

−4

0.445

0.1990.117

0.2930.020

0.7601.38

×10

−6

0.0410.800

0.5170.002

0.4770.005

0.085

0.019

VGF

VGF220-235

220 VPE

RAPL

PPSV

PSQFQ

235

AR-A

R3.04

×10

−73.68

×10

−6

0.864

0.1920.129

0.3940.002

0.9373.37

×10

−8

0.0740.626

0.4300.005

0.3570.023

0.073

0.022

VGF

Neuroendocrineregulatory

peptide-4

489 N

APP

EPVPP

PRAAPA

PTHV507

KK-R

S0.094

0.018

0.275

0.0960.429

0.2520.037

0.2640.035

−0.0520.734

0.4440.004

0.4960.002

0.076

0.017

VGF

VGF496-507

496 PPP

RAAPA

PTHV507

PV-RS

0.012

0.009

0.604

0.2830.035

0.3220.011

0.4160.002

0.0120.936

0.4500.003

0.4390.005

0.068

0.025

aThe

statisticalanalysisshow

sthat

BMAAinducedsignificant

changesin

theneuropeptid

elevelsin

theneonatalstriatum

comparedwith

maleandfemalevehiclecontrols.Three

linearmodelswere

employed

todetect

differencesin

relativepeptidelevelsbetweenthegroups.(i)In

thefirstmodel,the

summationof

groupmeans

formales

(fourgroups)was

considered

asfixedeffectsandindividual

samples

weredefinedasrandom

effects.T

hismodelprovides

thebestestim

ateofthetreatm

enteffecto

fBMAAinmales

only.(ii)

Inthesecond

model,the

summationofgroupmeans

forsex(twogroups),

thetreatm

entgroups

inboth

sexes(twogroups,control

andM/F150)

andtheinteractionbetweensexandtreatm

entwereconsidered

asfixedeffects.Individualsam

ples

weredefinedas

random

effects.

Thismodelconsiderstheinteractioneffectbetweenmales

andfemales

andtheeffectof

150mg/kg

BMAA,w

hich

istheonly

dose

measuredin

both

males

andfemales.(iii)

Inthethird

model,the

summationof

thegroupmeans

(sixgroups)forallgroupswas

considered

asfixedeffectsandindividualsamples

weredefinedas

random

effects,tocompare

allsixgroups,and

basedon

thismodel,the

varianceestim

ates

arepresentedforthebiological

(sam

ple)

andtechnicalbasedon

therepeated

measurements

ofthesamples

(with

in).

bAnF-test,followed

byT-testin

gwas

used

forintergroup

comparisonsofsinglepeptides.D

ifferenceswereconsidered

statisticallysignificant

atp<0.05

(markedinbold).Abbreviations

andcharacters:M

C=malevehiclecontrols,M

40=males

treatedwith

40mg/

kgBMAA,M

150=males

treatedwith

150mg/kg

BMAA,M

460=males

treatedwith

460mg/kg

BMAA,F

C=femalevehiclecontrols,F

150=females

treatedwith

150mg/kg

BMAA,Int

=Interaction

betweensexandtreatm

ent,log 2

=fold-changevalues

onlog 2

scale,p=p-values,V

ariancesample=biologicalvariancefrom

themodel,V

ariancewith

in=technicalreplicatevariancefrom

themodel.

Num

bers

with

inbracketsareused

todescrib

eshorterversions

ofpreviously

annotatedpeptides.Italic/boldtyping

indicatesnon-recognized

peptides.cPrecursors:7B

2=Neuroendocrineprotein7B

2;CCKN

=Cholecystokinin;CMGA=Chrom

ogranin-A;MCH

=Promelanin-concentratin

ghorm

one;PE

NK=Proenkephalin-A;SC

G1-3=Secretogranin1-3;

SMS=Somatostatin

;TBB2C

=Tubulin

beta-2C

chain;

TKN1=Protachykinin-1;

TYB10

=Thymosin

beta-10;

TYB4=Thymosin

beta-4;VGF=NeurosecretoryproteinVGF.



In contrast to chromogranin-A and secretogranin-1, allpeptides derived from the secretogranin-2 precursor werereduced in treated males compared with the vehicle controls,but the reduction of only two peptides was statisticallysignificant. YLNQEQAEQGREHLA (novel, SCG2 598−612) was reduced (0.82-fold) in the M460 group, and theone amino acid shorter peptide LNQEQAEQGREHLA(novel, SCG2 599−612) was dose-dependently reduced inthe M150 and M460 groups (0.86-fold and 0.81-fold,respectively). Both the M150 and M460 dose groupsdisplayed significantly reduced levels of the secretograninprecursor compared to vehicle control group. No treatmenteffect could be observed in the F150 group, but a statisticallysignificant interaction term for both of these peptides wasobserved (Table 1 and Supporting Information Figure 2). Inaddition, one peptide, ELSAERPLNEQIAEAE (novel, SCG338−57), derived from the secretogranin-3 precursor showeda dose-dependent increase in the BMAA-treated males(Table 1).

Both Increased and Reduced Levels of Peptides from theMCH, Somatostatin, and Cortistatin Precursors

The highest BMAA dose (460 mg/kg) exerted the largesteffect on the relative peptide levels in the striatum, andpeptides from some precursors were only affected in the M460group. Two MCH precursor peptides, the amidated form ofthe peptide neuropeptide-glutamic acid-isoleucine (NEI)EIGDEENSAKFPI(Amide) and EIGDEENSAKFPIG,showed a statistically significant increase in the M460 group(1.49 and 1.41-fold, respectively) (Figure 3). In contrast, theSANSNPAMAPRE (amino acid residues 1−12 of somatosta-tin-28) peptide from the somatostatin precursor was reduced(0.64-fold) in the M460 group compared with the vehiclecontrol group (Table 1).Furthermore, the peptide (pGlu)QERPPLQQPPHRD

from the cortistatin precursor was detected in all animals inthe M460 group and one animal in the M40 group, but notin any other group, indicating a selective increase of thisspecific peptide in BMAA-treated animals. The detectedpeptide was a shorter version (amino acid residues 1−13) of

Figure 1. Principal component analysis to visualize the neuropeptide analysis data of the striatum 24 h after the last treatment in neonatal male ratsadministered BMAA on PND 9−10. (A) The score plot summarizes the potential relationships between the individuals and separates the groups onboth axes. The corresponding loading (B) and contribution plot (C) show the peptides affected in male BMAA-treated rat pups. The largestcontribution was from peptides originating from the VGF precursor. The scale of the y-axis is presented in standard deviations. (D) A contribution plotof the sex/treatment-related effects in BMAA-treated rat pups on PND 9−10. The relative contribution of peptides from the TYB4 precursor reflectsdifferences in the peptide levels between the males and females. The scale of the y-axis is presented in standard deviations. Abbreviations: MC = malevehicle controls; M40 = males treated with 40 mg/kg BMAA; M150 = males treated with 150 mg/kg BMAA; M460 = males treated with 460 mg/kgBMAA; FC = female vehicle controls; F150 = females treated with 150 mg/kg BMAA. Precursors: VGF =Neurosecretory protein VGF; TBB2A/B/C =Tubulin beta-2A/B/C chain, SCG1−3, Secretogranin 1−3; PCSK1 = ProSAAS; MCH = Pro melanin-concentrating hormone; CMGA =Chromogranin-A; CCKN = Cholecystokinin; TYB10 = Thymosin beta-10; TYB4 = Thymosin beta-4; TKN1 = Protachykinin-1; PENK =Proenkephalin-A; PDYN = Proenkephalin-B; CBLN1 = Cerebellin-1; 7B2 = Neuroendocrine protein 7B2.



the known neuropeptide cortistatin-29, which has a dibasiccleavage site at the C-terminal end. In addition, significantly

lower levels of the tubulin beta-2C chain derived peptideSVVPSPKVSDTVVEPYNA were observed in M460 (0.81-fold)

Figure 2.The relative levels of nine neuropeptides from the VGF precursor in the neonatal striatum of BMAA-treatedmale and female rats. The baselineis control males (MC). (A) A dose-dependent increase of most peptides at 24 h after the last treatment was observed in neonatal male rats administeredBMAA on PND 9−10. The highest levels were observed among peptides in close proximity to the NERP-3 peptide. There was also a significant increaseof NERP-4 peptide in both males and females. The error bars display the 95% confidence interval. The y-axis is presented in log2 scale. Confidenceintervals not overlapping the baseline are statistically significant compared with control males. (B) An overview of the VGF precursor where thequantified peptides are outlined. Sequences marked in red are recognized peptides. (C) A summarized figure indicating that neonatal BMAA-exposureaffects the level of VGF on the precursor level. Confidence intervals not overlapping the baseline are statistically significant compared with control males.To facilitate the interpretation, the significant differences are marked with asterisks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with MC.Abbreviations: MC = male vehicle controls; M40 = males treated with 40 mg/kg BMAA; M150 = males treated with 150 mg/kg BMAA; M460 = malestreated with 460 mg/kg BMAA; FC = female vehicle controls; F150 = females treated with 150 mg/kg BMAA.

Figure 3.The relative levels of neuropeptide-glutamic acid-isoleucine and one additional peptide from theMCHprecursor in the neonatal striatum at 24h after the last treatment in male and female rats administered BMAA on PND 9−10. The baseline is control males (MC). A significant increase in therelative level of both peptides was observed in males treated with 460 mg/kg BMAA, whereas a nonsignificant increase in the neuropeptide-glutamicacid-isoleucine was observed in females treated with 150 mg/kg. The error bars display the 95% confidence interval. The y-axis is presented in log2 scale.Confidence intervals not overlapping the baseline are statistically significant compared with control males. To facilitate the interpretation, the significantdifferences are marked with asterisks. **p < 0.01, ***p < 0.001 compared with MC. Abbreviations: MC = male vehicle controls; M40 = males treatedwith 40 mg/kg BMAA; M150 = males treated with 150 mg/kg BMAA; M460 = males treated with 460 mg/kg BMAA; FC = female vehicle controls;F150 = females treated with 150 mg/kg BMAA.



compared with the controls. Consistently, the homologouspeptide SVMPSPKVSDTVVEPYNA derived from the tubulin

beta-2A/C chain showed the same trend, with a 0.84-foldreduction.

Figure 4. (A) The relative levels of 12 neuropeptides from the proenkephalin-A (PENK) precursor in the neonatal striatum at 24 h after the lasttreatment in male and female rats administered BMAA on PND 9−10. The baseline is control males (MC). An increased relative level of most peptideswas observed in the highest dose in males, but only the SPQLEDEAKE reached statistical significance. A similar increase was also observed for femaleswith several statistically significant changes compared with female controls. The response in females treated with 150 mg/kg BMAA was in many cases,as high or even higher than that of males treated with 460 mg/kg BMAA. (B) A summarized figure showing that neonatal BMAA-exposure affects thelevel of PENK on the precursor level. The error bars display the 95% confidence interval. The y-axis is presented in log2 scale. Confidence intervals notoverlapping the baseline are statistically significant compared with control males. To facilitate the interpretation, the significant differences are markedwith asterisks. *p < 0.05, **p < 0.01, ***p < 0.001 compared with MC. Abbreviations: MC =male vehicle controls; M40 =males treated with 40 mg/kgBMAA; M150 = males treated with 150 mg/kg BMAA; M460 = males treated with 460 mg/kg BMAA; FC = female vehicle controls; F150 = femalestreated with 150 mg/kg BMAA.



Treatment-Induced Sex-Dependent Changes of Peptidesfrom the Proenkephalin-A, Protachykinin-1, andProdynorphin Precursors

On the precursor level, proenkephalin-A demonstrated a sex-dependent BMAA-induced increase in the striatum of the F150group with no differences between the sexes when comparingmale and female vehicle control groups. The LEDEAKELQpeptide (amino acid residues 4−12 of propeptide 198−209)showed a statistically significant 1.56-fold increase in the F150group, compared with both male and female controls (p < 0.05)and close to significant at the overall level (ANOVA; p = 0.05).Furthermore, the full propeptide 198−209 SPQLEDEAKELQ,Met-enkephalin-Arg-Phe (MEAP) and the SPQLEDEAKEpeptides were significantly increased in the F150 groupcompared with female vehicle controls (Figure 4).Interestingly, a dose-dependent change in proenkephalin-A at

the precursor level was observed. The M40 group had asignificantly reduced relative level, followed by no changes in theM150 group and a significantly increased relative level in theM460 group. This was also observed as a clear trend in all twelvepeptides from the proenkephalin-A precursor (Table 1 andFigure 4). The most affected peptide SPQLEDEAKE (aminoacid residues 1−10 of propeptide 198−209) showed a significant1.49-fold increase in the M460 group compared with thecontrols.All three peptides from the protachykinin-1 precursor showed

the highest levels in the F150 group. No differences weredetected between the male and female controls. The relativelevels of the C-terminal-flanking peptide were significantlyincreased in the F150 group. Substance P and neurokinin Ademonstrated the same trend but the ANOVA was notsignificant. All three neuropeptides were significantly increasedin the F150 group compared with female controls (Figure 5).Two out of three detected peptides from the prodynorphinprecursor demonstrated the same trend as proenkephalin-A andprotachykinin 1-derived peptides. The prodynorphin derivedSQENPNTYSEDLDV (amino acid residues 15−28 of Leumor-phin) peptide was most affected after BMAA treatment andshowed a significant 1.22-fold increase in the F150 groupcompared with the control, but the ANOVA was not statisticallysignificant (ANOVA; p = 0.058) (Supporting InformationTable 1).

Treatment-Independent Sex Differences in Peptides fromThymosin-Beta Precursors

At the precursor level, thymosin beta-4 was reduced in bothBMAA-treated females and female controls compared with males(Supporting Information Figure 3). Moreover, the statisticsrevealed a significant 0.69-fold reduction in the relative levels ofthe PLPSKETIEQEKQAGES (amino acid residues 28−44 ofthymosin beta-4) peptide in females compared with males. Thefull thymosin beta-4 sequence with N-terminal acetylation alsoshowed a significant 0.81-fold decrease in the females comparedwith males (p < 0.05), which was not significant at the overalllevel (ANOVA; p = 0.072). The full-length peptide of thymosinbeta-10 showed the same trend (Supporting Information Figure 4).In addition, the PTKETIEQEKRSEIS peptide (amino acidresidues 30−44 of thymosin beta-10) from the thymosin beta-10precursor showed significantly reduced levels (0.75-fold) infemales compared with males. The data also revealed additionalpeptides with sex differences in the striatum; however, thiseffect was, in most cases, confounded by differences in thetreatment effects (e.g., peptides from 7B2, cerebellin-1 andsecretogranin-2).

■ DISCUSSION

We have previously shown that neonatal exposure to thecyanobacterial toxin BMAA (40 and 150 mg/kg) on PND 9−10impaired adult learning and memory function without anydistinct acute or long-term histopathological changes in thebrain, whereas a higher neonatal dose (460 mg/kg) induced celldeath and long-term neuronal degeneration in the hippocampus,without neuronal cell death in the striatum.18,21 BMAA is a mixedglutamate receptor agonist that may induce perturbations of theneurodevelopment as the glutamatergic system is involved in themodulation of many of the events that occur during braindevelopment.33 The present study demonstrated that exposureto noncytotoxic doses of BMAA on PND 9−10 inducedsignificant neuropeptide changes in the rat neonatal striatum,suggesting that neuropeptide profiling provides a sensitivemethod to characterize the impact of BMAA on brain function.The neuropeptide changes in the neonatal striatum generallyoccurred at the peptide precursor level, although there wereexceptions, indicating more complex mechanisms. Using a label-free mass spectrometry-based approach, a dose-dependent increasein the relative levels of several VGF-derived neuropeptides was

Figure 5. The relative levels of substance P, neurokinin A and the C-terminal flanking peptide from the protachykinin-1 (TKN1) precursor in theneonatal striatum at 24 h after the last treatment in male and female rats administered BMAA on PND 9−10. The baseline is control males (MC). Ansignificant increase in the relative level of all these peptides was observed in female rat pups treated with 150 mg/kg BMAA compared with controlfemales. No similar increase was observed in male rat pups treated with BMAA. The error bars display the 95% confidence interval. The y-axis ispresented in log2 scale. Confidence intervals not overlapping the baseline are statistically significant compared with control males. To facilitate theinterpretation, the significant differences are marked with asterisks. *p < 0.05, **p < 0.01 compared with MC. Abbreviations: MC = male vehiclecontrols; M40 =males treated with 40mg/kg BMAA;M150 =males treated with 150mg/kg BMAA;M460 =males treated with 460mg/kg BMAA; FC= female vehicle controls; F150 = females treated with 150 mg/kg BMAA.



demonstrated. A significant dose- and sex-dependent increase inthe relative level of peptides derived from the proenkephalin-Aprecursor and an increase in peptides derived from otherneuropeptide precursors, such as MCH, was also detected. Incontrast, the relative levels of peptides derived from precursors,such as the secretogranin-2, were dose-dependently reduced.The BMAA-induced effects on neuropeptide levels in theneonatal striatum suggest that this neurotoxin might induce aselective up- or downregulation of the of peptide precursors.However, it cannot be excluded that BMAA might also changethe processing of the peptide precursors through convertases andpeptidases to yield different levels of neuropeptides.

VGF and Brain Development

VGF is a developmentally regulated secretory peptide precursorexpressed by neurons, whose transcription and secretion areinduced through neurotrophins, such as BDNF, NGF and NT-3.34 VGF is a precursor for several biologically active peptides,such as NERP-4.34−36 The expression of VGF peaks during braindevelopment and is proteolytically processed during neuronaldifferentiation.37 In vitro studies have demonstrated that theinduction of VGF synthesis is closely associated with neuriteoutgrowth36 and axonal sprouting/synaptogenesis.38,39 Thepresent study revealed that neonatal BMAA treatment eliciteda significant and dose-dependent increase in the striatalexpression of three novel peptides and one recently publishedpeptide, namely, VGF 211−217, VGF 220−235, VGF 496−507,and NERP-4,32 respectively. The VGF precursor contains severalputative sites for proteolytic cleavage and undergoes cell-specificprocessing.38 Five additional peptides, including the previouslyknown VGF 180−194, also showed a significant increase.Furthermore, in the precursor, the peptides VGF 211−217, andVGF 220−235 are in close proximity to the bioactive NERP-3peptide.32 The VGF 211−217 peptide is conserved between rat,human and mouse and has a typical neuropeptide N-terminalcleavage site. Thus, we propose that several of the peptidesderived from the VGF precursor and induced by noncytotoxicdoses of BMAA in the neonatal striatummight be novel bioactivepeptides.The present study also revealed differential changes in the

levels of peptides from the chromogranin precursors chromog-ranin-A and secretogranin-1 in the striatum after neonatal BMAAexposure at the highest dose. Secretogranins, also calledchromogranins, are important for the neuroendocrine systemand have been implicated in the formation of large dense coresecretory vesicles, which are essential for functional synapses.40

Secretogranins are also precursors of peptides suggested topromote neuronal differentiation.40−42 In addition, we observedan increase of the MCH precursor-derived peptide NEI, whichhas been implicated in neuronal differentiation.43

Cholecystokinin and Neuroprotection

The present study also revealed a significant increase of thecholecystokinin-derived peptide QPVVPVEAVDPMEQ (part ofpropeptide 21−45) in the neonatal striatum of the rats in thehighest dose group (M460). Cholecystokinin is induced byglutamate,44 and cholecystokinin-derived peptides reportedlyprotect against glutamate-induced toxicity in cultured ratneurons45 suggesting that the increased cholecystokinin levelin the neonatal striatum of BMAA-treated rat pups might protectstriatal neurons, as no cell death was observed in the neonatalstriatum at this dose.18

Neuropeptides and BMAA-Induced Motor Changes

Neuropeptides, such as enkephalins, dynorphins, substance P,and neurokinin A and B, are present in the CNS before birth andexhibits significant developmental reorganization during the firstpostnatal weeks, accompanied by receptor-specific developmen-tal trajectories.46,47 For example, the enkephalin system issensitive to early life environmental manipulations.46 Thus, theseneuropeptides are involved in dopamine actions in the striatumand nucleus accumbens and have been implicated in the controlof both normal locomotion and stereotyped motor behaviorsresulting from imbalances within the nigrostriatal system.48 Forexample, the tachykinins have been implicated as agonists tomodulate motor activity in animal models of Parkinson’s disease.Furthermore, substance P-dependent mechanisms have beenimplicated in the regulation of muscle tone. In the striatum,substance P evokes the release of other transmitters, includingMet-enkephalin.47 The present study revealed an increased levelof enkephalins after neonatal BMAA exposure. This result couldbe important for the induction of acute but transient hyper-activity in neonatally exposed rats. The hyperactivity wasaccompanied with impaired motor function in the highest dosegroup (460 mg/kg).16 Thus, the dose-dependent motor effectsobserved in neonatal rats treated with BMAA might be inducedthrough the stimulation of the glutamatergic system and theincrease in enkephalins, which was observed in the highest dosegroup. Notably, at the precursor level proenkephalin-Ademonstrated an interesting dose-dependent pattern: a lowdose (M40) resulted in a reduced level, followed by no changesin M150 and an increased level in the highest dose group(M460).We also observed an increased level of the shorter version

(amino acid residues 1−13) of the neuropeptide cortistatin-29,derived from the cortistatin precursor. This peptide was almostexclusively detected in the highest dose group. The expression ofcortistatin in the CNS is relatively restricted, with highest levelsin cortex and hippocampus49 and only a few cortistatin-positivecells are usually detected in the striatum. The anticonvulsantcortistatin-29 reduces locomotor activity, but induces seizures athigher doses.49,50 Thus, the increased level of cortistatin mightcontribute to the convulsions observed in a few animals treatedwith a high dose (460 mg/kg) of BMAA.16

Sex-Related Changes in Striatal Neuropeptides

Sex differences in neuropeptide distribution in the brain havebeen sparsely investigated and little is known about sex-specificneuropeptide expression and regulation in the developingbrain.51 However, the sex-dependent effects on the mRNAexpression of prodynorphin and the substance P precursorpreprotachykinin in the striatum of adult mice have beendemonstrated.52 The present study demonstrated that femaleBMAA-treated pups treated with an intermediate dose(150 mg/kg) exhibited the highest relative levels of theprotachykinin 1-derived C-terminal-flanking peptide, substanceP and neurokinin A. Furthermore, the BMAA-treated femalesdisplayed increased relative levels of several proenkephalinderived peptides, including met-enkephalin, with the samemagnitude as the highest dose in males (460 mg/kg). The sametrend was observed for prodynorhin-derived peptides. Takentogether, this result indicates that female neonates might havea higher susceptibility toward BMAA than male neonates.Unfortunately, the effects of BMAA in the neonatal brain haveonly been reported for male rat pups,18 and the potential sex-related effects of BMAA on behavior or brain histopathology in



female rat pups remain elusive. There are for instance sexdifferences in the hypothalamic-pituitary-adrenal (HPA) axis53,54

that might be associated with the sex-related effects of BMAA, ascorticosterone regulates the striatal mRNA levels of proenke-phalin and protachykinin.48 Sex differences in the regulation ofproenkephalin by estrogen55 and sex chromosome-dependenteffects are also possible.52 In addition, the present studydemonstrated lower striatal levels of thymosin beta-4 andthymosin beta-10 in female controls compared with malecontrols during the neonatal period, consistent with a previousstudy demonstrating lower female serum levels of thymosin beta-4 compared with males in hypophysectomised neonatal rats.56

Taken together, the present results merit further studiesconcerning sex differences in neuropeptide expression and therole of neuropeptides during sexual differentiation in the brain.

■ CONCLUSIONSThe present analysis of endogenous neuropeptides revealedsignificant BMAA-induced early changes in the neonatal striatumfollowing noncytotoxic doses on PND 9−10. A dose-dependentincrease of VGF-derived peptides and a significant modulation ofmany peptides derived from other precursors were observed.Because several of these neuropeptides play a role in thedifferentiation and survival of neurons, many of the changes inthe neonatal striatum might contribute to BMAA-inducedbehavioral changes. Other neuropeptide changes suggest sex-related differences in the susceptibility toward BMAA-inducedeffects. For most of these peptides, the BMAA-induced changesoccur at the precursor level, although there were exceptions,indicating more complex mechanisms. Altogether, the datasuggest that BMAA-induced significant changes in the striatalneuropeptidome that may contribute to developmental changesin the neonatal brain. The present neonatal model is the onlyanimal model that displays significant behavioral effects afterBMAA exposure at a short-term systemic dose as low as 40 mg/kg. The observed BMAA-induced changes of neuropeptides inthe neonatal striatum in the absence of neuronal cell deathsuggest that neuropeptide profiling might provide a sensitivecharacterization of the noncytotoxic effects of BMAA in theneonatal brain.

■ ASSOCIATED CONTENT

*S Supporting Information

Relative levels of neuropeptides from the secretogranin 1 (SCG1),secretogranin 2 (SCG2), Thymosin beta-4 (TYB4), and Thymosinbeta-10 (TYB10) precursors in the neonatal striatum at 24 h afterthe last treatment in male and female rats administered BMAAon PND 9−10; list of peptides identified and relatively quantifiedin this study. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*Address: Uppsala University, Department of PharmaceuticalBiosciences, Box 591, SE-751 24 Uppsala, Sweden. E-mail:[email protected]. Fax: +46-18-4714253.

Author Contributions#O.K. and K.K. contributed equally to this study.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors would like to thank Ms. Raili Engdahl for technicalassistance. This work was financially supported through grantsfrom the Swedish Research Council FORMAS, the SwedishResearch Council, and the K&A Wallenberg Foundation.

■ REFERENCES(1) Banack, S. A.; Johnson, H. E.; Cheng, R.; Cox, P. A. Production ofthe neurotoxin BMAA by a marine cyanobacterium.Mar. Drugs 2007, 5(4), 180−196.(2) Cox, P. A.; Banack, S. A.; Murch, S. J.; Rasmussen, U.; Tien, G.;Bidigare, R. R.; Metcalf, J. S.; Morrison, L. F.; Codd, G. A.; Bergman, B.Diverse taxa of cyanobacteria produce beta-N-methylamino-L-alanine, aneurotoxic amino acid. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (14),5074−5078.(3) Brand, L. E.; Pablo, J.; Compton, A.; Hammerschlag, N.; Mash, D.C. Cyanobacterial blooms and the occurrence of the neurotoxin beta-N-methylamino-L-alanine (BMAA) in South Florida aquatic food webs.Harmful Algae 2010, 9 (6), 620−635.(4) Jonasson, S.; Eriksson, J.; Berntzon, L.; Spacil, Z.; Ilag, L. L.;Ronnevi, L. O.; Rasmussen, U.; Bergman, B. Transfer of a cyanobacterialneurotoxin within a temperate aquatic ecosystem suggests pathways forhuman exposure. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (20), 9252−9267.(5) Banack, S. A.; Cox, P. A. Biomagnification of cycad neurotoxins inflying foxes: implications for ALS-PDC in Guam. Neurology 2003, 61(3), 387−389.(6) Spencer, P. S.; Nunn, P. B.; Hugon, J.; Ludolph, A. C.; Ross, S. M.;Roy, D. N.; Robertson, R. C. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science1987, 237 (4814), 517−522.(7) Pablo, J.; Banack, S. A.; Cox, P. A.; Johnson, T. E.;Papapetropoulos, S.; Bradley, W. G.; Buck, A.; Mash, D. C.Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease.Acta Neurol. Scand. 2009, 216−225.(8) Nilsson, A.; Falth, M.; Zhang, X.; Kultima, K.; Skold, K.;Svenningsson, P.; Andren, P. E. Striatal alterations of secretogranin-1,somatostatin, prodynorphin, and cholecystokinin peptides in anexperimental mouse model of Parkinson disease. Mol. Cell. Proteomics2009, 8 (5), 1094−1104.(9) Rossbach, U.; Nilsson, A.; Falth, M.; Kultima, K.; Zhou, Q.;Hallberg, M.; Gordh, T.; Andren, P. E.; Nyberg, F. A quantitativepeptidomic analysis of peptides related to the endogenous opioid andtachykinin systems in nucleus accumbens of rats following naloxone-precipitated morphine withdrawal. J. Proteome Res. 2009, 8 (2), 1091−1098.(10) Scholz, B.; Alm, H.; Mattsson, A.; Nilsson, A.; Kultima, K.;Savitski, M. M.; Falth, M.; Skold, K.; Brunstrom, B.; Andren, P. E.;Dencker, L. Neuropeptidomic analysis of the embryonic Japanese quaildiencephalon. BMC Dev. Biol. 2010, 10, 30.(11) Scholz, B.; Skold, K.; Kultima, K.; Fernandez, C.; Waldemarson,S.; Savitski, M. M.; Soderquist, M.; Boren, M.; Stella, R.; Andren, P.;Zubarev, R.; James, P. Impact of temperature dependent samplingprocedures in proteomics and peptidomics - a characterization of theliver and pancreas post mortem degradome. Mol. Cell. Proteomics 2011,10 (3), M900229MCP200.(12) Karamyan, V. T.; Speth, R. C. Animal models of BMAAneurotoxicity: a critical review. Life Sci. 2008, 82 (5−6), 233−246.(13) Perry, T. L.; Bergeron, C.; Biro, A. J.; Hansen, S. [beta]-N-Methylamino-l-alanine: Chronic oral administration is not neurotoxic tomice. J. Neurol. Sci. 1989, 94 (1−3), 173−180.(14) Smith, Q. R.; Nagura, H.; Takada, Y.; Duncan, M. W. Facilitatedtransport of the neurotoxin, beta-N-methylamino-L-alanine, across theblood-brain barrier. J Neurochem 1992, 58 (4), 1330−7.(15) Karlsson, O.; Berg, C.; Brittebo, E. B.; Lindquist, N. G. Retentionof the cyanobacterial neurotoxin beta-N-methylamino-l-alanine inmelanin and neuromelanin-containing cellsa possible link between



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mailto:[email protected]

Parkinson-dementia complex and pigmentary retinopathy. Pigm. CellMelanoma Res. 2009, 22 (1), 120−130.(16) Karlsson, O.; Lindquist, N. G.; Brittebo, E. B.; Roman, E. Selectivebrain uptake and behavioral effects of the cyanobacterial toxin BMAA(beta-N-methylamino-L-alanine) following neonatal administration torodents. Toxicol. Sci. 2009, 109 (2), 286−295.(17) Karlsson, O.; Berg, A. L.; Lindstrom, A. K.; Arnerup, G.; Roman,E.; Bergquist, J.; Hanrieder, J.; Lindquist, N. G.; Brittebo, E.; Andersson,M. Neonatal exposure to the cyanobacterial toxin BMAA induceschanges in protein expression, and neurodegeneration in adulthippocampus. Toxicol. Sci. 2012, 130 (2), 391−404.(18) Karlsson, O.; Roman, E.; Berg, A. L.; Brittebo, E. B. Earlyhippocampal cell death, and late learning and memory deficits in ratsexposed to the environmental toxin BMAA (beta-N-methylamino-L-alanine) during the neonatal period. Behav. Brain Res. 2011, 219 (2),310−320.(19) Myhrer, T. Neurotransmitter systems involved in learning andmemory in the rat: a meta-analysis based on studies of four behavioraltasks. Brain Res. Brain Res. Rev. 2003, 41 (2−3), 268−287.(20) Devan, B. D.; Hong, N. S.; McDonald, R. J. Parallel associativeprocessing in the dorsal striatum: segregation of stimulus-response andcognitive control subregions. Neurobiol. Learn. Mem. 2011, 96 (2), 95−120.(21) Karlsson, O.; Roman, E.; Brittebo, E. B. Long-term cognitiveimpairments in adult rats treated neonatally with beta-N-methylamino-L-alanine. Toxicol. Sci. 2009, 112 (1), 185−195.(22) Lalonde, R.; Strazielle, C. Brain regions and genes affectingpostural control. Prog. Neurobiol. 2007, 81 (1), 45−60.(23) Timiras, P. S.; Sherwood, N. M. A Stereotaxic Atlas of theDeveloping Rat Brain; University of California Press: Berkeley, LosAngeles. CA, 1970.(24) Skold, K.; Svensson, M.; Norrman, M.; Sjogren, B.; Svenningsson,P.; Andren, P. E. The significance of biochemical and molecular sampleintegrity in brain proteomics and peptidomics: stathmin 2−20 andpeptides as sample quality indicators. Proteomics 2007, 7 (24), 4445−4456.(25) Svensson, M.; Skold, K.; Svenningsson, P.; Andren, P. E.Peptidomics-based discovery of novel neuropeptides. J Proteome Res.2003, 2 (2), 213−219.(26) Falth, M.; Skold, K.; Norrman, M.; Svensson, M.; Fenyo, D.;Andren, P. E. SwePep, a database designed for endogenous peptides andmass spectrometry. Mol. Cell. Proteomics 2006, 5 (6), 998−1005.(27) Falth, M.; Skold, K.; Svensson, M.; Nilsson, A.; Fenyo, D.;Andren, P. E. Neuropeptidomics strategies for specific and sensitiveidentification of endogenous peptides.Mol. Cell. Proteomics 2007, 6 (7),1188−1197.(28) Craig, R.; Beavis, R. C. TANDEM: matching proteins withtandem mass spectra. Bioinformatics 2004, 20 (9), 1466−1467.(29) Kultima, K.; Nilsson, A.; Scholz, B.; Rossbach, U. L.; Falth, M.;Andren, P. E. Development and evaluation of normalization methodsfor label-free relative quantification of endogenous peptides. Mol. Cell.Proteomics 2009, 8 (10), 2285−2295.(30) Pinheiro, J.; Bates, D.; DebRoy, S.; Sarkar, D.; The R Core Teamnlme: Linear and Nonlinear Mixed Effects Models; R Foundation forStatistical Computing; Vienna, Austria, 2009.(31) R Development Core Team. R: A Language and Environment forStatistical Computing; R Foundation for Statistical Computing: Vienna,Austria, 2011.(32) Sasaki, K.; Takahashi, N.; Satoh, M.; Yamasaki, M.; Minamino, N.A peptidomics strategy for discovering endogenous bioactive peptides. J.Proteome Res. 2010, 9 (10), 5047−5052.(33) Ruediger, T.; Bolz, J. Neurotransmitters and the development ofneuronal circuits. Adv. Exp. Med. Biol. 2007, 621, 104−15.(34) Snyder, S. E.; Pintar, J. E.; Salton, S. R. Developmental expressionof VGF mRNA in the prenatal and postnatal rat. J. Comp. Neurol. 1998,394 (1), 64−90.(35) Salton, S. R.; Fischberg, D. J.; Dong, K. W. Structure of the geneencoding VGF, a nervous system-specific mRNA that is rapidly and

selectively induced by nerve growth factor in PC12 cells.Mol. Cell. Biol.1991, 11 (5), 2335−249.(36) Benson, D. L.; Salton, S. R. Expression and polarization of VGF indeveloping hippocampal neurons.Dev. Brain Res. 1996, 96 (1−2), 219−228.(37) Cocco, C.; D’Amato, F.; Noli, B.; Ledda, A.; Brancia, C.;Bongioanni, P.; Ferri, G. L. Distribution of VGF peptides in the humancortex and their selective changes in Parkinson’s and Alzheimer’sdiseases. J. Anat. 2010, 217 (6), 683−693.(38) Snyder, S. E.; Cheng, H. W.; Murray, K. D.; Isackson, P. J.;McNeill, T. H.; Salton, S. R. The messenger RNA encoding VGF, aneuronal peptide precursor, is rapidly regulated in the rat centralnervous system by neuronal activity, seizure and lesion. Neuroscience1998, 82 (1), 7−19.(39) Lombardo, A.; Rabacchi, S. A.; Cremisi, F.; Pizzorusso, T.; Cenni,M. C.; Possenti, R.; Barsacchi, G.; Maffei, L. A developmentallyregulated nerve growth factor-induced gene, VGF, is expressed ingeniculocortical afferents during synaptogenesis. Neuroscience 1995, 65(4), 997−1008.(40) Mahata, M.; Mahata, S. K.; Fischer-Colbrie, R.; Winkler, H.Ontogenic development and distribution of mRNAs of chromogranin Aand B, secretogranin II, p65 and synaptin/synaptophysin in rat brain.Dev. Brain Res. 1993, 76 (1), 43−58.(41) Chen, M.; Tempst, P.; Yankner, B. A. Secretogranin I/chromogranin B is a heparin-binding adhesive protein. J. Neurochem.1992, 58 (5), 1691−1698.(42) Fischer-Colbrie, R.; Kirchmair, R.; Kahler, C.M.;Wiedermann, C.J.; Saria, A. Secretoneurin: a new player in angiogenesis and chemotaxislinking nerves, blood vessels and the immune system. Curr. Protein Pept.Sci. 2005, 6 (4), 373−385.(43) Kistler-Heer, V.; Schlumpf, M.; Lichtensteiger, W. Melanocortinand MCH precursor-derived NEI effects on striatum-midbrain co-cultures. Peptides 1998, 19 (8), 1317−1327.(44) Akaike, A.; Tamura, Y.; Sato, Y.; Ozaki, K.; Matsuoka, R.; Miura,S.; Yoshinaga, T. Cholecystokinin-induced protection of culturedcortical neurons against glutamate neurotoxicity. Brain Res. 1991, 557(1−2), 303−307.(45) Tamura, Y.; Sato, Y.; Akaike, A.; Shiomi, H. Mechanisms ofcholecystokinin-induced protection of cultured cortical neurons againstN-methyl-D-aspartate receptor-mediated glutamate cytotoxicity. BrainRes. 1992, 592 (1−2), 317−325.(46) Nylander, I.; Roman, E. Neuropeptides as mediators of the early-life impact on the brain; implications for alcohol use disorders. Front.Mol. Neurosci. 2012, 5, 77.(47) Page, N. Brain Tachykinins. In Handbook of Biologically ActivePeptides; Kastin, J., Ed. Elsevier, Inc.: Burlington, MA, 2006; pp 763−769.(48) Angulo, J. A.; McEwen, B. S. Molecular aspects of neuropeptideregulation and function in the corpus striatum and nucleus accumbens.Brain Res. Brain Res. Rev. 1994, 19 (1), 1−28.(49) de Lecea, L.; del Rio, J. A.; Criado, J. R.; Alcantara, S.; Morales, M.;Danielson, P. E.; Henriksen, S. J.; Soriano, E.; Sutcliffe, J. G. Cortistatinis expressed in a distinct subset of cortical interneurons. J. Neurosci.1997, 17 (15), 5868−80.(50) Wilson, D. N.; Chung, H.; Elliott, R. C.; Bremer, E.; George, D.;Koh, S. Microarray analysis of postictal transcriptional regulation ofneuropeptides. J. Mol. Neurosci. 2005, 25 (3), 285−298.(51) Rugarn, O.; Hammar, M.; Theodorsson, A.; Theodorsson, E.;Stenfors, C. Sex differences in neuropeptide distribution in the rat brain.Peptides 1999, 20 (1), 81−86.(52) Chen, X.; Grisham, W.; Arnold, A. P. X chromosome numbercauses sex differences in gene expression in adult mouse striatum. Eur. J.Neurosci. 2009, 29 (4), 768−776.(53) Rhodes, M. E.; Rubin, R. T. Functional sex differences (’sexualdiergism’) of central nervous system cholinergic systems, vasopressin,and hypothalamic-pituitary-adrenal axis activity in mammals: a selectivereview. Brain Res. Brain Res. Rev. 1999, 30 (2), 135−152.(54) DeSantis, S. M.; Baker, N. L.; Back, S. E.; Spratt, E.; Ciolino, J. D.;Moran-SantaMaria, M.; Dipankar, B.; Brady, K. T. Gender differences in



the effect of early life trauma on hypothalamic-pituitary-adrenal axisfunctioning. Depression Anxiety 2011, 28 (5), 383−392.(55) Segarra, A. C.; Acosta, A. M.; Gonzalez, J. L.; Angulo, J. A.;McEwen, B. S. Sex differences in estrogenic regulation of preproenke-phalinmRNA levels in themedial preoptic area of prepubertal rats. BrainRes. Mol. Brain Res. 1998, 60 (1), 133−139.(56)Wise, T.; Klindt, J.; Macdonald, G. J.; Ford, J. J. Effects of neonatalsexual differentiation, growth hormone and testosterone on thymicweights and thymosin-beta 4 in hypophysectomized rats. J. Reprod.Immunol. 1991, 19 (1), 43−54.



neurotoxin-induced neuropeptide perturbations in striatum of neonatal rats

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