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Journal of Psychiatric Research 53 (2014) 166e172

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Journal of Psychiatric Research

journal homepage: www.elsevier .com/locate/psychires

Increased plasma levels of BDNF and inflammatory markersin Alzheimer’s disease

Mayara Chaves Faria a,b, Gisele Santos Gonçalves a,b, Natália Pessoa Rocha c,Edgar Nunes Moraes d,e, Maria Aparecida Bicalho d,e, Marco Túlio Gualberto Cintra d,Jonas Jardim de Paula d, Luís Felipe José Ravic de Miranda d,Alessandro Clayton de Souza Ferreira f, Antônio Lúcio Teixeira c,e, Karina Braga Gomes a,Maria das Graças Carvalho a,b, Lirlândia P. Sousa a,b,*

aDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazilb Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av.Antonio Carlos, 6627,Pampulha, 31270-901 Belo Horizonte, Minas Gerais, Brazilc Laboratório Interdisciplinar de Investigação Médica, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, BrazildAmbulatório de Idosos do Instituto Jenny de Andrade Faria do Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte,Minas Gerais, BrazileDepartamento de Clínica Médica da Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazilf Instituto Hermes Pardini, Belo Horizonte, Minas Gerais, Brazil

a r t i c l e i n f o

Article history:Received 7 October 2013Received in revised form19 December 2013Accepted 30 January 2014

Keywords:NeurotrophinsNeurotrophic factorsNeurodegenerationInflammationBiomarkersBrain derived neurotrophic factorsTNFR1

* Corresponding author. Programa de Pós-GraduaçãFaculdade de Farmácia, Universidade Federal de Min6627, Pampulha, 31270-901 Belo Horizonte, Minas GeTel.: þ55 31 34096883; fax: þ55 3134092651.

E-mail address: lipsousa72@gmail.com (L.P. Sousa

http://dx.doi.org/10.1016/j.jpsychires.2014.01.0190022-3956/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. Neurotrophic factors andinflammatory markers may play considerable roles in AD. In this study we measured, through Enzyme-Linked Immunosorbent Assay, the plasma levels of brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) and neuronal growth factor (NGF), as well as tumor necrosisfactor-alpha soluble receptors, sTNFR1 and sTNFR2, and soluble intercellular adhesion molecule 1(sICAM-1), in 50 AD patients, 37 patients with mild cognitive impairment (MCI) and 56 healthy elderlycontrols. BDNF levels, expressed as median and interquartile range, were higher for AD patients (2545.3,1497.4e4153.4 pg/ml) compared to controls (1503.8, 802.3e2378.4 pg/ml), P < 0.001. sICAM-1 was alsohigher in AD patients. sTNFR1 levels were increased in AD when compared to controls and also to MCI.GDNF, NGF and sTNFR2 levels showed no significant differences among the studied groups. The increasein BDNF might reflect a compensatory mechanism against early neurodegeneration and seems to berelated to inflammation. sTNFR1 appears to mark not only the inflammatory state but also differentiatesbetween MCI and AD, which may be an additional tool for differentiating degrees of cognitiveimpairment.

� 2014 Elsevier Ltd. All rights reserved.

1. Objectives of the study and background

AD is characterized by the presence of extracellular amyloidplaques, containing deposits of beta-amyloid protein (Ab), intra-cellular neurofibrillary tangles and extensive neuronal and synapticloss (Adlard and Cummings, 2004). The genetic background plays aconsiderable role in AD as polymorphisms in the gene of

o em Ciências Farmacêuticas,as Gerais, Av.Antonio Carlos,rais, Brazil.

).

apolipoprotein E (APOE) affect the risk of the late-onset form of thedisease, with the ε4 allele and the ε2 allele being respectivelyrelated to an increased and decreased risk for AD development(Leduc et al., 2010). Additionally, inflammatory processes followAlzheimer’s neuropathology (Papassotiropoulos et al., 2001). Acti-vatedmicroglia and astrocytes identified near amyloid plaques leadto increased levels of inflammatory cytokines as: interleukin-1 (IL-1), interleukin-3 (IL-3) and tumor necrosis factor a (TNF-a)(Akiyama et al., 2000; Song et al., 2009).

Levels of TNF-a soluble receptors (sTNFR1 and sTNFR2) havealso been described as representatives of the inflammatory activityin AD patients and individuals with mild cognitive impairment(MCI) (Diniz et al., 2010). sTNFR1 and 2 are induced by TNF-a in

M.C. Faria et al. / Journal of Psychiatric Research 53 (2014) 166e172 167

humans, and are more stable than the cytokine itself. Therefore,these soluble receptors reflect the levels of TNF-a over a prolongedtime (Buchhave et al., 2010). TNF-a can increase the expression ofadhesion molecules such as the vascular cell adhesion protein 1(VCAM-1) and the intercellular adhesion molecule 1 (ICAM-1) inthe endothelium (Collins et al., 1995). These molecules are bio-markers of microvascular lesion and the plasma levels of theirsoluble forms are reported to be increased in AD (Breteler, 2000;Ewers et al., 2010).

Recent findings have highlighted changes in the levels of neu-rotrophic factors in post-mortem AD brain, where increased levelsof neuronal growth factor (NGF) and decreased levels of brainderived neurotrophic factor (BDNF) were reported (Ferrer et al.,1999; Hellweg et al., 1998; Hock et al., 2000). BDNF was, howev-er, strongly positive in damaged neurites surrounding amyloidplaques (Ferrer et al., 1999), suggesting a positive regulatory cir-cuitry of this neurotrophic factor in damaged brain areas.

The ability of neurotrophic factors to provide support, growthand survival to neurons, brings the idea that a loss or dysfunction intheir production might be related to the development of neuro-degenerative disorders (Siegel and Chauhan, 2000). In effect, BDNFis able to decrease the amount of Ab and generate peptides whichhave neurotrophic and neuroprotective roles (Rohe et al., 2009;Thornton et al., 2006).

Changes in peripheral levels of neurotrophic factors have, aswell, been observed in the context of psychiatric or neurode-generative diseases. Serum BDNF was found to be reduced(Forlenza et al., 2010; Lee et al., 2009) in AD and MCI patients, incontrast with its increase reported by Angelucci et al. (2010) andLaske et al. (2006). Schizophrenia (SZ) and bipolar disease (BD)patients also displayed elevated BDNF circulating levels (Gamaet al., 2007; Barbosa et al., 2012). Measurements of glial cell-derived neurotrophic factor (GDNF) in blood showed its elevationin euthymic BD patients (Barbosa et al., 2011) and AD ones(Straten et al., 2009), while opposite findings have also beendescribed for the latter subjects (Marksteiner et al., 2011).Regarding NGF, it was proposed that the pre-dementia stage thatprecedes AD is marked by a decrease in its serum levels (Schaubet al., 2002). Such decrease was also observed in Huntington andParkinson disease patients (Lorigados Pedre et al., 2002; Tassetet al., 2012).

BDNF is upregulated at the vicinity of Ab plaques where animmune response is evoked by activated glial cells (Burbach et al.,2004; Ferrer et al., 1999). The implications of microglial activationand infiltrating cytokines in neuronal survival and memory pro-cesses in the context of neuronal damage illustrate the connectionbetween the immune and nervous systems (Yirmiya and Goshen,2011). Immune cells can produce the majority of neurotrophicfactors and cytokines are able to influence such production(Kerschensteiner et al., 2003). Indeed, TNF-a and IL-6 enhanced theproduction of BDNF by cultured monocytes (Schulte-Herbrüggenet al., 2005) while a strong activation of the immune system wasassociated with a downregulation of BDNF mRNA expression(Lapchak et al., 1993). Reinforcing the cited modulatory circuitry,such downregulation was abrogated when cultured astrocyteswere exposed to IL-4 (Derecki et al., 2010). Additionally, immunecells can express receptors for neurotrophic factors, which could beinvolved in the regulation of the inflammatory response (Nockherand Renz, 2006; Siegel and Chauhan, 2000). As the involvementof inflammation in AD pathology has been reported (Akiyama et al.,2000), the role of neurotrophic factors in the central nervous sys-tem along with their possible role in the inflammatory setting,makes them conceivable biomarkers for AD.

Heterogenous results concerning levels of neurotrophic factorshave been reported so far, mainly due to differences in analyzed

biological specimen (brain tissue, serum, plasma) and the diseasestage inwhich it is sampled. As homogeneity between the studies ispursued, and similar evaluation protocols are applied to the sub-jects, more comparable results will be obtained enabling a betterunderstanding of neurotrophic factors as biomarkers for AD.

The progressive and irreversible characteristics of AD haveprompted researchers to look for useful biomarkers for either itsearly diagnosis or for understanding the course of the disease. Inthe past two decades, blood (serum, plasma or circulating cells) hasbeen considered a potential source of biomarkers for the diagnosisof AD and for research purposes as it is easily obtained compared tocerebral spinal fluid (CSF) (Caramelli et al., 2011).

In the present study we measured the plasma levels of BDNF,GDNF, NGF as well as sTNFR1, sTNFR2 and sICAM-1 in order toevaluate the inflammatory and neurotrophic profile in AD and MCIpatients in comparison with controls.

2. Methods

2.1. Subjects

This study evaluated 56 healthy controls, 37 patients with MCIand 50 AD patients, recruited from the outpatient clinic of InstitutoJenny de Andrade Faria, Universidade Federal de Minas Gerais(UFMG), Brazil. The groups were matched for age which rangedfrom61 to 89 years old. None of the participants were in use of anti-inflammatories.

The diagnosis of AD and MCI was based on screening testsincluding the Mini-Mental State Examination (MMSE) (Folsteinet al., 1975) with cut-off limits adjusted according to the educa-tion level, CERAD Protocol (Morris et al., 1989), Activities of DailyLiving Scale (Katz et al., 1963), Instrumental activities of daily living(Pfeffer et al., 1982) and Hachinski Ischemic Scale (<2) (Rosen et al.,1980). The diagnosis of probable AD was performed according tothe DSM-IV and NINCDS-ADRDA criteria (McKhann et al., 2011).

No familial cases of AD were included in this study. The diag-nosis of amnestic MCI followed the recommendation of Petersen(2004). The recruited patients underwent a thorough geriatricassessment, including medical history, physical and laboratorialscreening tests, brain imaging exams and neuropsychological tests,when necessary.

The individuals enrolled in the control group were cognitivelyintact, with no personal and family (first degree relatives) history ofneuropsychiatric diseases. They were submitted to clinical tests toexclude other psychiatric disorders.

In order to exclude patients or controls with acute inflammatoryprocess, C-Reactive Protein (CRP) level was measured in all in-dividuals through highly sensitive Near Infrared Particle Immuno-assay rate methodology (IMMAGE� Immunochemistry systems,Beckman Coulter, Galway, Ireland). The elected participants hadlevels under 10 mg/L (Ansar and Ghosh, 2013). In effect, evenwhenCRP is increased due to the time course of mild cognitive impair-ment, it does not reach values as high as 10 mg/L (Karim et al.,2013).

Demographic and clinical data of the participants were obtainedretrospectively through medical record review or through inter-view during the collection of the blood samples.

AD patients were divided into two different groups according totheir Clinical Dementia Rate (CDR) score (Morris, 1993). CDR1corresponds to a mild stage of dementia, and 27 patients werefound in this group. 16 patients were at a moderate stage of de-mentia and 4were at a severe stage, theywere listed together in theCDR2/CDR3 group.

This study was approved by the Ethics Committee of Uni-versidade Federal de Minas Gerais, and the participants signed an

Table 1Clinical characteristics of the AD, MCI and control groups of participants.

Controls(n ¼ 56)

MCI(n ¼ 37)

AD(n ¼ 50)

P

Age (SD) 74.3 (5.7) 73.1 (7.2) 76.2 (5.7) 0.054Gender (W/M) 43/13 20/17 33/17 0.072Education (IR) 11 (4.5e14.5) 5 (4e8) 3.5 (1e4.3) 0.001*aec

Antidepressants 14.3% 16.2% 54% <0.001*a,b

AChEI e e 73.2%

MCI, mild cognitive impairment; AD, Alzheimer’s disease; SD, standard deviation;IR, interquartile range; W, women, M, men; APOE ε4, presence of the ε4 apolipo-protein E allele; AChEI, acetylcholinesterase inhibitors.*Significant P-value (a ¼ 0.05). a, Significant for control � AD; b, significant forMCI � AD; c, significant for control � MCI.

M.C. Faria et al. / Journal of Psychiatric Research 53 (2014) 166e172168

informed consent after the research was explained to them. Con-sent was only given by the participants who fulfilled the criteriaand presented a MMSE score compatible to a non-demented state.Otherwise, consent was obtained from relatives or caregivers.

2.2. Measurement of neurotrophic factors, sTNFRs and sICAM

Samples of 5 mL of blood were collected in tubes with EDTA andcentrifuged, after which they were aliquoted and frozen at �80 �Cuntil the analysis was performed.

The BDNF, GDNF, NGF, sTNFR1 and sTNFR2 and sICAM-1 plasmaconcentrations were measured using an ELISA kit according to themanufacturer’s instructions (R&D Systems�, Minneapolis, USA).Concentrations were obtained against a standard curve calibratedwith known amounts of protein and expressed as pg/mL, except forsICAM-1 levels, which were expressed as ng/mL. As previousstudies have demonstrated the impact of APOE genotype oninflammation parameters (Jofre-Monseny et al., 2007), we analyzedsTNFR1 and sTNFR2 plasma levels of the participants according totheir genotype.

2.3. Statistical analysis

Descriptive statistics were used to report demographic andclinical characteristics of the participants. Association betweennominal variables was assessed with Pearson’s chi-square test.

Normality of distribution was tested for quantitative variablesby the ShapiroeWilk test and all data were non-normally distrib-uted. Comparisons among the experimental groups were done byKruskaleWallis test followed by a ManneWhitney test and Bon-ferroni correction, when appropriate. Results were expressed inmedian and interquartile range due to the non-normal distributionof the studied variables. Spearman’s test was performed to examinethe correlation between two variables. A forward stepwise multi-variate logistic regression analysis was used to identify the inde-pendent predictors of MCI and AD among the variables with a P-value <0.2 in the univariate analysis. HosmereLemeshow test was

Table 2Plasma levels (pg/mL) GDNF, NFG, sTNFR2 and sICAM-1 (ng/mL) in controls, MCI and AD

Control MCI

Median IQR Median

GDNF 559 25e712 588NGF 53 42e110 56sTNFR2 6330 3001e8773 6239sICAM-1 273 223e324 297

MCI, mild cognitive impairment; AD, Alzheimer’s disease; GDNF, Glial cell-derived neursoluble receptor 2; sICAM-1, soluble intercellular adhesion molecule 1.*Significant P-value (a ¼ 0.05). a, Significant for control � AD.

applied to evaluate the adequacy of the multivariate model. Allstatistical tests were two-tailed and were performed using a sig-nificance level of a ¼ 0.05 and an adjusted significance level ofa ¼ 0.017 when Bonferroni correction was applied. Statistical an-alyses were performed using SPSS software version 17.0 (SPSS Inc.,Chicago, IL, USA).

3. Results

The clinical characteristics of the participants can be observed inTable 1. Out of 37 MCI patients, 19 were classified as single domainamnestic, 9 were multiple domain amnestic MCI and 9 were notclassified.

The median and interquartile range values for the plasma levels(pg/mL) of GDNF, NGF, sTNFR2 and sICAM-1 in the groups (controls,MCI and AD) are shown in Table 2. Plasma levels of GDNF, NGF, andsTNFR2didnot showsignificantdifferences among the threegroups.

AD patients presented higher plasma levels of BDNF (2545.3,1497.4e4153.4 pg/ml) compared to controls (1503.8, 802.3e2378.4 pg/ml), P < 0.001. MCI patients presented a median of1866.9, 888.9e5724.7 pg/ml for BDNF levels, which althoughhigher than the levels for the control group, did not reach signifi-cance after Bonferroni correction (P ¼ 0.045). Although the BDNFlevels for the AD group presented a highermedian than the ones forMCI subjects, there was not a significant difference between thetwo values (P ¼ 0.224) (Fig. 1a).

Regarding the dementia stage of AD, BDNF plasma levels inCDR1 patients (3018.0, 1516.6e5132.8 pg/mL) and CDR2/CDR3 pa-tients (3425.8, 1864.9e5380.6 pg/mL) were not statisticallydifferent, P ¼ 0.644. sTNFR1 values also did not differ between theCDR groups (CDR1 ¼ 2020.1, 1509.0e2473.4 pg/mL; CDR2/CDR3 ¼ 2088.7, 1463.4e3126.3 pg/mL). Similarly, no differenceswere observed for sICAM-1 levels according to the degree ofseverity of AD (CDR1 ¼ 338.7, 283.3e441.2 ng/mL; CDR2/CDR3 ¼ 393.6, 277.9e487.4 ng/mL).

Since studies have shown that antidepressants and acetylcho-linesterase inhibitors (AChEI) can interfere in BDNF levels (Leyheet al., 2008) we tested if it could be demonstrated in our studiedpopulation.

BDNF plasma levels did not vary between the participants ac-cording to the use of antidepressants: 2268.7, 818.9e5164.7 pg/mLin individuals under antidepressant treatment and 1660.1, 1021.2e4037.3 pg/mL in the ones who were not in use of antidepressants(P ¼ 0.490). There was no difference between its levels in non-treated AD patients (1593.1, 872.8e3394.5 pg/mL) and the ones inuse of acetylcholinesterase inhibitors (AChEI) (3007.7, 1775.7e5172.2 pg/mL), P ¼ 0.222.

sTNFR1 concentrations were significantly increased in the ADpatients (2019.9, 1485.9e2537.2 pg/ml) compared to the othergroups (Controls: 1292.7,1032.7e1812.7pg/ml;MCI: 1291.8,1025.2e1526.5 pg/ml), P < 0.001 for each comparison, as shown in Fig. 1b.

patients. Data are shown as median and interquartile range.

AD P-value

IQR Median IQR

66e700 501 12e741 0.29342e105 57 50e99 0.269

3552e7886 7838 4447e11987 0.301226e363 353 285e459 0.008*a

otrophic factor; NGF, neuronal growth factor; sTNFR2, tumor necrosis factor-alpha

Fig. 1. BDNF and sTNFR1 plasma concentrations in elderly controls, MCI and AD. Themiddle line represents the median. A: ***P < 0.001, for controls � AD. B: ***P < 0.001for controls � AD and MCI � AD. MCI, mild cognitive impairment; AD, Alzheimer’sdisease; BDNF, Brain derived neurotrophic factor; sTNFR1, tumor necrosis factor-alphasoluble receptor 1.

Fig. 2. sTNFR1 plasma levels among different APOE genotypes. The middle line rep-resents the median. *P < 0.05 and **P < 0.01. sTNFR1, tumor necrosis factor-alphasoluble receptor 1.

Table 3Multivariate logistic regression model applied separately to control and MCI pa-tients, controls and AD group and MCI and AD patients.

Variables Categories P-value CI 95% OR OR

Control � MCIBDNF Higha 0.021* 1.20e9.25 3.33APOE ε4 carrier <0.001* 2.16e15.24 5.73Control � ADBDNF Higha <0.001* 2.13e14.60 5.57APOE ε4 carrier 0.012* 1.32e9.39 3.52sTNFR1 Highb 0.001* 1.83e12.70 4.82MCI � ADsTNFR1 Highb <0.001* 3.20e28.82 9.6

MCI, mild cognitive impairment; AD, Alzheimer’s disease; BDNF, Brain derivedneurotrophic factor; APOE, apolipoprotein E; OR, Odds ratio.*Significant P-value (a ¼ 0.05).

a >75th percentile of control group: 2378.4 pg/mL.b >75th percentile of control group: 1812.7 pg/mL.

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sTNFR1 plasma levels were higher for ε4 allele carriers (1537.8,1322.4e2448.0 pg/mL) compared to individuals with the ε3ε3 ge-notype (1397.8, 1040.0e1921.4 pg/mL; P ¼ 0.011), and ε2ε2 or ε2ε3APOE genotype (1092.9, 946.4e1426.9 pg/mL; P ¼ 0.002) (Fig. 2).The participants with ε2ε4 genotype were excluded in this analysisdue to the opposite effect of these alleles. No significant differenceswere found among the genotype groups regarding BDNF levels(P ¼ 0.785).

sICAM-1 plasma levels were significantly higher in AD patientscompared to controls, P ¼ 0.008 (Table 2). AD patients’ sICAM-1levels were higher than the ones for MCI patients, however, afterBonferroni correction such difference was not statistically signifi-cant, P ¼ 0.041.

In AD patients, sTNFR1 was positively correlated with sICAM-1(r ¼ 0.449 and P ¼ 0.004). When considering all the three stud-ied groups together, it was observed a positive correlation betweensTNFR1 and BDNF (r ¼ 0.170; P ¼ 0.043) and sTNFR1 and age(r ¼ 0.268; P ¼ 0.001). No other age-related correlations wereobserved with the analyzed variables.

We also analyzed the variables using a multivariate logisticregression model. Higher BDNF levels (>75th percentile of controlgroup: 2378.4 pg/mL), and the presence of the ε4 allele of the APOEgenewere independently associated with MCI when the MCI groupwas compared to controls. Higher levels of BDNF and sTNFR1(>75th percentile of control group: 1812.7 pg/mL) as well as the ε4allele were independently associated with Alzheimer’s diseasewhen controls and AD patients were considered for the analysis.And when analyzing the results of MCI and AD patients, sTNFR1

emerged as an independent variable highly associated to Alz-heimer’s disease (Table 3).

4. Discussion

In this study, BDNF and sTNFR1 plasma levels were increased inAD patients versus controls, being the latter parameter alsoincreased in these patients compared to MCI ones. Also, a positivecorrelation was observed for sTNFR1 and sICAM-1 in the AD group.

Whilst there is enough evidence supporting a crucial role forBDNF in several cognitive processes (Linnarsson et al., 1997; Leckieet al., 2012), the alterations in its levels and how the body managesthe challenges in neuronal homeostasis are not easily understood.

Most of the studies performed in post-mortem brains, either bymeasuring BDNF’s mRNA or protein content, reported decreasedlevels for AD patients (Phillips et al., 1990; Ferrer et al., 1999). A fewother studies showed that BDNF levels or the expression of its re-ceptor was increased in the hippocampus of AD patients (Duranyet al., 2000; Kao et al., 2012).

Concerning the peripheral levels of BDNF, less agreement isfound. Laske et al. (2007) as well as Lee et al. (2009) reported thatBDNF was decreased in the serum of AD patients. The first did notfind correlation between serum and CSF levels, and the later alsofound decreased levels for MCI patients but admitted uncertainties

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concerning the characterization of the dementia rate of thesubjects.

In keeping with our results, increased BDNF circulating levels inMCI and AD patients were observed by Angelucci et al. (2010) andLaske et al. (2006); the first, similarly to our findings, showed thatthis increase was not related to treatment with AChEI or antide-pressants. Differences in the methodology used to classify MCIpatients and the inclusion of amnestic-single domain andamnestic-multiple domain patients in different proportions in theMCI groups could be a possible explanation for inconsistent resultsregarding the levels of BDNF for individuals with mild cognitiveimpairment (Borba, 2012).

After observing that astroglial responses against Ab occur beforeobvious neuronal damage can be detected (Kimura et al., 2004),Kimura et al. (2006) found that BDNF was rapidly produced byastrocytes in opposition to the deleterious effects of Ab. Based onthat, they stated that such phenomenon possibly occurs due to acompensatory mechanism that would happen in the initial stagesof dementia. The increase in BDNF in vivo could represent anattempt to rescue neurons from damage and diminish the amyloidburden since it was able to reverse the toxic effect of Ab in vitro(Arancibia et al., 2008).

Laske et al. (2006) proposed that peripheral levels of BDNFwould increase in the early stages of dementia and decrease ac-cording to the severity of the neurodegeneration. They observedhigher BDNF serum levels in early-stage AD patients (MMSEscore > 21) compared to controls and also found that BDNF wasdecreased in later-stages (MMSE score < 21).

After the AD group of our study was divided into subgroups(CDR1 and CDR2/CDR3), the number of patients in each of thembecame rather small, limiting the possibility of observing a changein its levels according to the stage of the disease. The same limi-tation might have impacted the analysis regarding sTNFR1 andsICAM-1 within the different CDR groups. Therefore new studieswith a larger number of patients carefully divided into subgroupscharacterized by different dementia rates should be performed.

As there is still controversy concerning the correlation betweenBDNF levels in CNS and circulation (Klein et al., 2011; Elfving et al.,2010) more studies should be encouraged in order to confirm suchcorrelation. It may also be that the time course of central and pe-ripheral BDNF changes significantly differs, partially explaining thecontroversies.

Concerning the sTNFR1 receptor, we found that its plasmalevels were increased in AD patients compared to controls and toMCI patients, demonstrating a more prominent inflammatorystate in AD. Although there is not much information about therole of sTNFR1 and sTNFR2 as biomarkers in AD, Diniz et al.(2010) demonstrated that upon follow-up, MCI patients whodeveloped AD had higher sTNFR1 serum levels at baseline thanthe ones who remained as MCI, and through Cox regression theyverified that high serum sTNFR1 levels predicted the conversionfrom MCI to AD. Similarly to our study, they did not find signif-icant differences concerning TNF-a and sTNFR2 levels among thegroups of patients with AD, MCI and controls. In harmony withthose findings, the present study revealed, through logisticregression with the results of MCI and AD patients, that highsTNFR1 levels were strongly associated with Alzheimer’s disease.When only controls and AD patients were considered, sTNFR1was also independently associated with the disease. Diez-Ruizet al. (1995) have already mentioned that TNF-a soluble re-ceptors (sTNFRs) appeared to have more discriminative valuethan classic parameters for staging illnesses. If similar resultsconcerning sTNFR1 are found as more studies are performed, thissoluble receptor could eventually be used as a tool for differen-tiating degrees of cognitive impairment.

The validity of sTNFR1 as a marker of the inflammatory profile isfurther supported by the fact that it positively correlated withsICAM-1 expression. Increased expression of TNFR1 in the cellmembrane results in increased responsiveness to TNF-a mediatedICAM-1 expression (Cook et al., 2008). If such phenomenon impactsthe circulating levels of sTNFR1 and sICAM-1, it might explain thecorrelations found.

sTNFR1 was positively correlated with the age of the partici-pants. It is known that as age increases, a gradual loss of adaptiveimmunity takes place while mononuclear cells of innate immunityhave their activity increased. In such phenomenon, known asinflammaging, higher amounts of TNF-a, and consequently theirsoluble receptors, are produced (Giunta et al., 2008).

The positive correlation between BDNF and the inflammatorymarkers sTNFR1 is supported by the current knowledge that im-mune cells are able to produce this neurotrophin after several in-flammatory processes. The immune system can respond to aninjury by induction of BDNF, which has been proven bioactive andable to support neuronal survival in vitro (Kerschensteiner et al.,1999). Therefore, it is reasonable to imagine that in the beginningof neurodegeneration, the injury to the neurons and the inflam-mation scenario would be related to an increased production ofBDNF, as if the body were trying to compensate that damage (Laskeet al., 2006). Later, as the neurodegeneration proceeds, and Abplaques and tangles become more numerous, BDNF’s productionwould then be hindered (Murer et al., 1999; Garzon andFahnestock, 2007; Poon et al., 2011).

The APOE genotype is another characteristic that should betaken into account when discussing about inflammation and AD(Jofre-Monseny et al., 2007). In our study, the group of individualswho had the ε3ε4 and ε4ε4 APOE genotype displayed the highestlevels of plasma sTNFR1. Zhu et al. (2012) showed that immunecells from APOE4 knock-in mice were more activated after lipo-polysaccharide injection and themice displayed a greater andmoreprolonged increase of IL-1b, IL-6, TNF-a in their brains. A pro-spective study with human subjects showed that the use of non-steroidal anti-inflammatory drugs only reduced the risk for ADdevelopment in individuals who were APOE ε4 carriers (Szekelyet al., 2008). Therefore, the genetic background seems to play arole in how the immune cells respond to injuries and that responseis able to alter several parameters of measurement.

In spite of the expectations regarding NGF’s use as a therapeuticagent in AD for maintaining cholinergic neurons, the results ob-tained so far concerning its use as a peripheral marker have notshown enough consistency. Similarly to our results, Murase et al.(1991) and Serrano-Sanchez et al., 2001 did not find significantdifferences between NGF levels of AD patients and controls.

A better characterization of peripheral levels of GDNF in AD isdesirable. While our study shows no significant differences be-tween GDNF plasma levels in AD and control groups, results fromother researches showing its elevation or its decrease in AD pa-tients can also be found (Marksteiner et al., 2011; Straten et al.,2009).

At the current time point of Alzheimer’s research, whentherapeutic intervention is being thoroughly studied, it is rele-vant to understand the course of the disease in a molecular leveland specially focus on pre-dementia stages such as the MCI.This paper provides a wide panorama of peripheral molecularchanges in AD and MCI as it evaluates different neurotrophicfactors and inflammatory markers together. As a limitation ofour study, we highlight the lack of use of pathophysiologicalbiomarkers based on amyloid imaging and CSF measurementsfor the diagnosis of MCI and AD patients. It is known that thesebiomarkers increase the sensitivity and specificity of the diag-nosis (Lista et al., 2013). Since MCI is usually a very heterogenous

M.C. Faria et al. / Journal of Psychiatric Research 53 (2014) 166e172 171

entity, it is desirable to have this group more rigorously selected,and identify the patients that are more prone to develop AD. Ourpatients, though, underwent a thorough geriatric assessment,neuroimaging and neuropsychological assessment protocol (dePaula et al., 2013).

In order to evaluate the specificity of our findings on AD pa-tients, it would be interesting to perform the analysis described inthis study in patients with other dementias.

In conclusion, our findings indicate that AD patients have higherlevels of peripheral BDNF possibly due to a compensatory mecha-nism to fight early neurodegeneration or to an activation of im-mune cells. sTNFR1 appears to be an interesting peripheral markerof the inflammatory state as well as the impairment in cognition.sICAM-1 also marks an exacerbated inflammatory state in AD pa-tients and is correlated to sTNFR1 levels.

Role of the Funding Source

This work was supported by grants from Fundação de Amparo aPesquisa do Estado de Minas Gerais (FAPEMIG, Brazil), ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq,Brazil), and Pró-Reitoria de Pesquisa da Universidade Federal deMinas Gerais-PRPq, Brazil (Programa de Auxílio à Pesquisa deDoutores Recém-Contratados).

Contributors

Mayara Chaves Faria: Master’s student responsible for all essaysand elaboration of MSc dissertation.

Gisele Santos Gonçalves: PhD student involved patientsrecruitment, blood collection, many laboratory assays, statisticalanalysis and text writing.

Natália Pessoa Rocha: PhD students who has assisted the authorwith ELISA assays.

Edgar NunesMorais: MD, geriatriciane participation in patientsrecruitment by applying neuropsychiatric test.

Maria Aparecida Bicalho: MD, geriatrician e Participation inpatients recruitment and in the elaboration of the study design anddevelopment.

Jonas Jardim de Paula: PhD, neuropsychologist e participationin patients recruitment by applying neuropsychiatric test.

MarcoTúlio Gualberto Cintra: MD, geriatriciane participation inpatients recruitment and meetings concerning the study designand inclusion and exclusion criteria definition.

Luís Felipe Jose Ravic de Miranda: MD, geriatrician responsiblefor obtaining clinical data of the participants. Participation inmeeting concerning the research project as a whole.

Alessandro Clayton de Souza Ferreira: PhD, responsible for somelab assays and text revision.

Antônio Lúcio Teixeira: MD, neurologist and psychiatrist,participation in several meetings and discussions regarding thestudy design, providing suggestions and corrections in the presentmanuscript.

Karina Braga Gomes: PhD, responsible for genetic analysis, partof the statistical analysis and text revision.

Maria das Graças Carvalho: PhD e supervision of the researchproject and follow-up of the entire process, including writing.

Lirlândia Pires de Sousa: PhD e supervision of the researchproject and follow-up of the entire process, including writing.

Conflict of Interest

All other authors declare that they have no conflicts of interest.

Acknowledgment

We are very grateful to all participants and their relatives orcaregivers who kindly provided the necessary information for thisstudy.

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