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S100B as a Biomarker or Ca2+Signaling Dysregulation inSchizophreniaRicardo Sinz-Fuertes, Abdul Hye, Sarah Westwood, Alan Tunniclie, David HayesRicardo Sinz-Fuertes, LMS MSc MRCPsych, Kings College London

Dr. Sinz-Fuertes is a Locum Consultant in Community Forensic Psychiatry at The South London and

Maudsley Trust and is currently fnishing a Ph.D. working under the supervision o Proessor Sir Robin

Murray and Proessor Simon Lovestone. Dr. Sinz-Fuertes is analyzing protein expression in plasma

samples o patients suering rom schizophrenia using proteomics techniques such as two-dimensional

gel electrophoresis (2-PAGE) and mass spectrometry (LC/MS/MS).

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Introduction

Schizophrenia (ragmented mind or SCZ) has

traditionally been considered a neurodevelopmental

disorder1, but recent studies have uncovered evidence

o neurodegeneration in SCZ patients2.

Structural eatures consistently ound in SCZ brains

include an overall reduction in brain volume, thinning o

grey matter, ventricle enlargement, and smaller temporallobe structures such as the hippocampus, amygdala and

superior temporal gyri. Additional structural ndings

include a smaller prerontal cortex, thalamus, anterior

cingulate and corpus callosum3. Patients with SCZ also

show abnormal cerebral symmetry4, which is inversely

correlated to early age o onset and hemispheral

dominance. Some o these changes appear in the

early, prodromal stages o the disorder and are stable

throughout the course o the illness. However, in some

cases, there seems to be a neurodegenerative component

to the progression o these anatomical alterations. These

changes are also seen to a lesser extent in other psychoticdisorders such as Bipolar Aective Disorder (BPAD) and in

unaected relatives, all o which prevent these changes

rom being considered pathognomonic or diagnostically

relevant5. Moreover, although most o these changes are

present at illness onset, antipsychotic medication has

been shown to induce structural changes in basal ganglia

by increasing its volume (as with typical antipsychotics)6

or by decreasing it (as with atypical antipsychotics),

while increasing the thalamus and cortical grey matter

volumes7.

Post-mortem studies o the brain in individuals with SCZ

have shown lower overall brain weight and alterations

in brain symmetry. Additionally, enlarged ventricles and

structural changes in hippocampal, rontal and temporal

lobe regions, thinning o grey matter in the absence o

gliosis, abnormal neuronal migration, dysunctional

apoptosis, and synaptic and dendritic alterations are

common8-12.

In the brain, calcium is undamental in the control o

synaptic activity and memory ormation, a process that

leads to the activation o specic calcium-dependent

signal transduction pathways. Furthermore, dysregulation

in calcium homeostasis can lead to neurodegeneration13.

S100 proteins are a amily o low molecular weight

(1012 kDa) Ca2+binding proteins that are involved

in cellular processes such as contraction, motility,

cell growth and dierentiation, cell cycle progression,

transcription, structural organization o membranes,

dynamics o cytoskeleton constituents, protection rom

oxidative cell damage, protein phosphorylation and

secretion14. One S100 gene, S100B, is located in

chromosome 21q22, an area that has been linked to

SCZ and BPAD with psychosis15, 16. S100 proteins are

present exclusively in vertebrates17

.

S100B is a recognized biomarker or traumatic brain

injury (TBI) and several chronic neurological disorders

(SCZ, bipolar disorder, Alzheimers disease). In addition, it

may have utility as a biomarker in other disease states,

such as cerebral palsy, Downs syndrome, multiple

sclerosis and metastatic melanoma. S100B is the most

abundant member o the S100 amily expressed in the

brain, and is predominantly synthesized and secreted by

astrocytes. Increased extracellular levels o S100B in the

brain can result in neurodegeneration (Figure 1), are

indicative o neuronal injury or underlying chronic

infammation and can subsequently be measured in the

peripheral circulation, ater passing through the blood-

brain barrier. Measurement o S100B in blood is more

convenient than measuring cerebrospinal fuid (CSF),

since routine blood tests are almost invariably conducted

on admission to the hospital in psychiatric patients

whereas lumbar punctures or CSF analysis are extremely

rare in these settings.

In this study, we have used an ELISA that was designed

to measure S100B in plasma, serum and CSF. Uniquely,S100B can be determined in both heparin and EDTA

plasma samples. The ability to use EDTA plasma samples

enabled this study to be perormed utilizing archived

materials, without the need to collect resh serum

or heparin plasma samples as required by other

methodologies to determine S100B levels.

Here, we present a retrospective study o samples collected

rom SCZ patients, along with age-matched controls, on

the relationship between levels o S100B ound in the

peripheral circulation and disease occurrence.

Figure 1.

Mechanisms by which S100B can promote neurodegeration.When high levels o S100B are secreted by astrocytes,neurodegeneration can result, either by direct activationo reactive oxygen species (ROS) production and oxidativestress, or by indirect stimulation o microglia, causingsecretion o proapoptotic cytokines. (Image adapted rom

Sorci G. et al., Cardiovascular Psychiatry and Neurology,2010; vol. 2010, Article ID 656481, 13 pages.doi:10.1155/2010/656481).

Microgliaactivation

NeuronROS production, death

Astrocyte

S100B(high)

S100B(high)

IL-6TNF-a

NOTNF-a

NOIL-1bTNF-a

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Materials and Methods

Subjects. Patients aged 1865 years with a clinical

diagnosis o psychosis who presented or the rst

time to in-patient units o the South London and

Maudsley Mental Health National Health Service

(NHS) FoundationTrust between 2005 and 2008 were

approached or inclusion in the study. Written consent

was obtained. Cases with a diagnosis o organicpsychosis, learning disability, head injury, or with poor

command o the English language were excluded. Those

who metICD10 criteria or a diagnosis o psychosis

codes F20F29and F30F3318 were invited to participate

in the study. Controls were recruited over the same

period o time rom the local communities where the

cases lived. Potential control subjects were excluded

i they met criteria or psychosis, either by means o

reporting past psychiatric history o a psychotic disorder

or by being identied as possibly having one by the

PsychosisScreening Questionnaire19.

Samples were collected using a 23G butterfy needle

and Vacutainer needle holder ollowing standard

venipuncture in the cubital region, in the area o

anastomosis between the radial and the ulnar veins or

in the brachial vein, slightly proximal to this area. To

avoid coagulation o the sample, a 9 mL K3EDTA tube

(Greiner Bio-One, Cat. No. 455036X, lavender lid) was

used. The sample was immediately kept at 4 C until

being processed, with the time between collection and

processing o the sample never exceeding two hours.

The tube was labeled with the subject barcode (no

phenotypic inormation was indicated). The sample

was then centriuged at 3000 rc or 8 minutes at 4 C.The plasma was aliquoted into 0.5 mL eppendor tubes

and immediately rozen at -80 C.

Kit Design.EMD Millipores Human S100B ELISA kit

(Cat. No. EZHS100B-33K) was designed to enable

researchers to measure levels o S100B in CSF, serum or

plasma (Heparin or EDTA). This kit was validated with

human samples, but the assay is cross-reactive with

multiple species, including bovine, hamster, horse,

monkey, mouse, pig, rabbit and rat. The antibody pair

used in this assay measures S100B specically, and has

no cross-reactivity with S100A1, S100A6 and S100A13.

Assay Procedure.The ELISA was perormed in a 96-well

plate according to the instructions supplied with the

Human S100B ELISA Kit. All plasma samples were used as

neat (no prior dilution was required). Briefy, the plate

was rinsed with 300 L diluted wash buer. Subsequently,

50 L o controls and standards were added to the

appropriate wells. 50 L o plasma samples were added

to the appropriate wells, and 50 L o assay buer alone

were added to the blank wells. The plate was sealed and

incubated or 2 hours on an orbital shaker at room

temperature (RT).

The plate was washed 5 times with 300 L wash buer.

100 L detection antibody was added to all wells and

incubated or 1.5 hours on an orbital shaker at RT. Again,

the plate was washed 5 times with 300 L wash buer.

100 L enzyme solution was added and incubated or

30 minutes at RT on a shaker. Once more the plate was

washed 5 times with 300 L wash buer. 100 L o

substrate solution was added to all o the wells, and

incubated or 5-25 minutes depending on color change.

The reaction was stopped with 100 L stop solution and

absorbance was read in a plate reader set at 450 nM.

Results and Discussion

To examine the expression levels o S100B, we utilized

the EMD Millipore Human S100B ELISA. This ELISA

enabled the detection o S100B in plasma samples with

excellent sensitivity with a mean coecient o variation

(CV) o 7.35% between duplicates. The inter-assay CV was

calculated rom the provided QC1 and QC2 values across

6 plates. The mean inter-assay CV was 9.2%.

Figure 2 shows a representative standard curve rom the

Human S100B ELISA. All standard curves were generated

using a 5-parameter logistic (5-PL) regression t (SigmaPlot,

SYSTAT Sotware Inc.) with a linear range rom S7-S5

(666.67 74.07 pg/mL).

Quantied data rom SigmaPlot was entered into IBM

SPSS (v15), and a Shapiro-Wilk test or normality was

perormed (p>0.05), indicating the data was not normally

distributed or S100B. We thereore used the

nonparametric test (MannWhitney).

Figure 2.

A representative standard curve rom the Human S100BELISA (Cat. No. EZHS100B-33K).

Absorbance(450nm)

S100B Concentration (pg/mL)

0

0 2.74 8.23 24.69 74.07 222.22 666.67

0.2

0.4

0.6

0.8

0.1

0.3

0.5

0.7

Human S100B ELISA Standard Curve

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We compared the mean levels between the two groups

(control n=81 and SCZ n=80). The average level o S100B

in the control group was 40.91 pg/mL and the average

level in the SCZ group was 90.65 pg/mL. A substantially

elevated level o S100B protein was observed in the

peripheral circulation o the SCZ group compared to

the controls and this elevation was highly signicant

(Mann-Whitney, p=0.003, Figure 3).

This retrospective study using archived samples conrmed

previous ndings that S100B levels in peripheral blood

are signicantly higher than age-matched controls in

SCZ. Using this kit to measure S100B in easy-to-obtain

serum/plasma samples will improve patient compliance in

clinical studies, and reduce the requirement to draw CSF,

which has several associated risks.

Figure 3.Plasma rom SCZ patients shows higher expression levelso S100B compared to control subjects. S100B wasmeasured in plasma using the Human S100B ELISA(Cat. No. EZHS100B-33K). The sample set was split intotwo groups (control n=81, SCZ n=80). A non-parametrictest (Mann-Whitney) was applied to compare mean valuesbetween both groups. A signifcant dierence is observedin the mean values between control and the SCZ group(p=0.003, error bars indicate the standard error o the mean).

Reerences

1. Lewis, S.W., Murray, R.M. Obstetric complications,

neurodevelopmental deviance, and risk o schizophrenia.

J Psychiatr. Res 1987; 21 (4): 413-21.

2. Prez-Neri, I., et al. Possible mechanisms o neurodegeneration

in schizophrenia. Neurochem. Res. 2006, October 31; (10):

1279-94.

3. Keshavan, M. S., et al. Schizophrenia,just the acts: what

we know in 2008 Part 3: neurobiology. Schizophr. Res. 2008;

106: 89-107.

4. Crow, T. J., et al. Schizophrenia as an anomaly o development

o cerebral asymmetry. A postmortem study and a proposal

concerning the genetic basis o the disease. Arch. Gen.

Psychiatry. 1989; 46: 1145-1150.

5. Stoll, A. L., et al. Neuroimaging in bipolar disorder: what have

we learned? Biol. Psychiatry, 2000; 48: 505-517.

6. Navari, S., Dazzan, P. Do antipsychotic drugs aect brain

structure? A systematic and critical review o MRI ndings.

Psychol. Med. 2009; 39: 1763-1777.

7. Scherk, H., Falkai, P. Eects o antipsychotics on brain

structure. Curr. Opin. Psychiatry. 2006; 19:145-150.

8. Brown, R., et al. Postmortem evidence o structural brain

changes in schizophrenia. Dierences in brain weight,temporal horn area, and parahippocampal gyrus compared

with aective disorder. Arch. Gen. Psychiatry. 43: 36-42.

9. Harrison, P. J. The neuropathology o schizophrenia. A critical

review o the data and their interpretation. Brain. 1999; 122

(Pt 4): 593-624.

10. Iritani, S. Neuropathology o schizophrenia: a mini review.

Neuropathology. 2007; 27: 604-608.

11. Selemon, L. D. Goldman-Rakic, P. S., The reduced neuropil

hypothesis: a circuit based model o schizophrenia. Biol.

Psychiatry. 1999; 45: 17-25.

12. Selemon, L. D., et al. Smaller rontal gray matter volume in

postmortem schizophrenic brains. Am. J. Psychiatry. 2002;

159: 1983-1991.

13. Marambaud, P., et al. Calcium signaling in neurodegeneration.

Mol. Neurodegener. 2002; 4: 20.

14. Santamaria-Kisiel, L., et al. Calcium-dependent and

independent interactions o the S100 protein amily.

Biochem. J., 2006; 396: 201-214.

15. Liu, J., et al. SNPs and haplotypes in the S100B gene reveal

association with schizophrenia. Biochem. Biophys. Res.

Commun. 2005; 328: 335-341.

16. Roche, S., et al. Candidate gene analysis o 21q22: support

or S100B as a susceptibility gene or bipolar aective

disorder with psychosis. Am. J. Med. Genet. B.

Neuropsychiatr. Genet. 2007; 144B: 1094-1096.

17. Sedaghat, F., Notopoulos, A. S100 protein amily and its

application in clinical practice. Hippokratia. 2008; 12:198-204.

18. World Health Organization. The ICD10 Classication o

Mental and Behavioural Disorders, Clinical Description and

Diagnostic Guidelines. WHO, 1992.

19. Bebbington P.E., Nayani, T. The Psychosis Screening

Questionnaire. Int. J. Methods Psychiatr. Res. 1995; 5: 11.

Acknowledgments

The authors would like to thank the participants rom the

GAP study at the Institute o Psychiatry, Kings College

London, as well as the clinical team or sample collection.

This study was unded by the MRC, Alzheimers Research

UK and NIHR Biomedical Research Centre or Mental

Health. We are very grateul to EMD Millipore or

providing the Human S100B ELISAs.

ConcentrationofS100BinHumanPlasma(pg/mL)

0

120.00

100.00

80.00

60.00

40.00

20.00

SchizophreniaControl

S100B Expression Levels in Study Groups

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