Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer's disease

Neurobiology of Aging 27 (2006) 1763–1768

Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer’s disease Daniela Galimberti a,∗ , Chiara Fenoglio a , Carlo Lovati b , Eliana Venturelli a , Ilaria Guidi a , Barbara Corr`a a , Diego Scalabrini a , Francesca Clerici b , Claudio Mariani b , Nereo Bresolin a , Elio Scarpini a a

Department of Neurological Sciences, “Dino Ferrari” Center, University of Milan, IRCCS Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milan, Italy b Department of Neurology, University of Milan, Ospedale L. Sacco, Milan, Italy

Received 17 June 2005; received in revised form 21 September 2005; accepted 18 October 2005 Available online 22 November 2005

Abstract Upregulation of a number of chemokines, including monocyte chemotactic protein-1 (MCP-1), is associated with Alzheimer’s disease (AD) pathological changes. Emerging evidence suggests that inflammatory events precede the clinical development of AD, as cytokine disregulation has been observed also in patients with mild cognitive impairment (MCI). MCP-1 levels were evaluated in serum samples from 48 subjects with MCI, 94 AD patients and 24 age-matched controls. Significantly increased MCP-1 levels were found in MCI and mild AD, but not in severe AD patients as compared with controls. mRNA levels in peripheral blood mononuclear cells (PBMC), evaluated by quantitative RT-PCR analysis, paralleled serum MCP-1 levels. Moreover, a progressive MCP-1 decrease was observed over a 1-year follow up in a subgroup of MCI subjects converted to AD. MCP-1 upregulation is likely to be a very early event in AD pathogenesis, by far preceding the clinical onset of the disease. Nevertheless, as MCP-1 is likely to play a role in several pathologies with an inflammatory component, a possible usfulness as an early AD biomarker would be possible only in combination with other molecules. © 2005 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease (AD); Mild cognitive impairment (MCI); Monocyte chemoattractant protein-1 (MCP-1); Biomarker

1. Introduction Alzheimer’s disease (AD) is the most common form of dementia in the elderly. Amyloid beta (A␤) deposition in the brain is likely to play a crucial role during AD development as it originates a chronic inflammatory response, which likely contributes to neuronal death (see [1] for review). In this regard, immunoreactivity for a number of chemokines, as well as for their related receptors, has been demonstrated in resident cells of the CNS, and upregulation of some of them is associated with Alzheimer’s disease (AD) pathological changes [38]. Therefore, chemokines have been supposed to play a relevant role in the pathogenesis of inflammatory ∗

Corresponding author. Tel.: +39 02 55033858; fax: +39 02 50320430. E-mail address: [email protected] (D. Galimberti).

0197-4580/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2005.10.007

processes occurring during the development of the pathology, mainly because of their chemotactic activity on brain phagocytes [19]. So far, microglial cells cultured in vitro have been shown to display an increased migratory response upon treatment with beta chemokines, including monocyte chemotactic protein-1 (MCP-1), suggesting that these molecules may play an important role in the trafficking of mononuclear phagocytes within the brain [29]. Lu et al. [18] disrupted MCP-1 gene and tested MCP-1-deficient mice in models of inflammation, demonstrating a severe impairment in monocyte trafficking, suggesting that MCP-1 is uniquely essential for the recruitment of these cells [18]. However, in spite of its chemotactic effect, MCP-1 expression alone does not cause inflammatory activation of cells, but contributes to enhance the inflammatory response upon treatment with other stimuli [8,13]. MCP-1 has been found immunohistochemically

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in mature, but not in immature, senile plaques and in reactive microglia of brain tissues from patients with AD. This observation lead to the hypothesis that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques [16]. However, previous evidence suggests that inflammatory responses might also promote neuroprotection, as in the case of the complementderived C5a anaphylatoxin [25]. Recently, MCP-1, together with IP-10 and IL-8 levels, have been evaluated in cerebrospinal fluid (CSF) from a group of 21 patients with AD as compared with age-matched controls [9]. Both MCP-1 and IL-8 CSF levels were higher in all AD patients, whereas IP-10 levels were increased only in a subgroup of patients, and correlated negatively with cognitive decline, measured with the mini-mental state examination (MMSE), suggesting a role of chemokines in the early stages of the disease, in which inflammation is likely to be more pronounced [9]. Besides CSF studies, peripheral MCP-1 levels have been evaluated in serum, but no significant differences were found in AD patients as compared with age-matched controls [7,9]. MCP1 is likely to be influenced by age, as a correlation between age and increased MCP-1 levels has been observed in the Japanese population [15]. Emerging evidence suggests that intrathecal inflammation precedes the clinical development of Alzheimer’s disease, as cytokine disregulation has been observed in patients with mild cognitive impairment (MCI), which can be defined as an isolated deficit in memory [26] often associated with decline to AD [22]. In particular, CSF increased production of the proinflammatory cytokine TNF␣, together with a decreased production of the anti-inflammatory cytokine TGF␤ in CSF from subjects with MCI has been shown [33]. In a recent study, both serum and CSF inflammatory-related molecules, including MCP-1, were proposed as candidate biomarkers for monitoring the inflammatory process in AD [32]. However, with regard to a possible use of inflammatory molecules to predict evolution from MCI to AD, no investigation on MCP-1 has been so far carried out, despite a growing body of evidence supporting the hypothesis that some peripheral biochemical modifications occur very early during AD pathogenesis. In particular, in serum samples from subjects with MCI there is evidence of an oxidative imbalance, including an increase in plasma total homocysteine (tHcy) levels and a decrease in total antioxidant capacity (TAC). The same modifications were observed in AD patients as well [12].

These data strongly suggest that a peripheral modification of inflammatory factors and molecules related to oxidative stress occurs early during AD development, when the clinical manifestation of the disease is not already clearly defined. To further study the role of MCP-1 during AD pathogenesis, we evaluated its levels in serum samples from individuals with MCI, which is considered the very early stage of AD [22], as well as in a large group of AD patients, either in a mild/moderate or severe phase of the disease, and compared them with those of age-matched subjects with no neurological diseases (CON). In addition, MCP-1 mRNA levels were determined by quantitative RT-PCR analysis in peripheral blood mononuclear cells (PBMC).

2. Materials and methods 2.1. Subjects Ninety-four Italian AD patients (70 women and 24 men, mean age at disease onset 75.0 ± 0.71 years) as well as 48 subjects with MCI (29 women and 19 men, mean age at onset 73.3 ± 0.90) were consecutively recruited at Alzheimer Units of Ospedale Maggiore Policlinico (Milan) and Ospedale L. Sacco (Milan). All patients underwent a standard battery of examinations, including medical history, physical and neurological examination, screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT) and, if indicated, positron emission computed tomography (PET). Dementia severity was assessed by the clinical dementia rating (CDR) and the minimental scale examination (MMSE). Disease duration was defined as the time in years between the first symptoms (by history) and the clinical diagnosis. The diagnosis of probable AD was made by exclusion according to NINCDS-ADRDA criteria [21]. Twelve patients had an early disease onset (EOAD; ≤65 years), whereas the remaining subjects had a late onset of the disease (LOAD; >65 years). MCI diagnosis was made in accordance with Petersen et al.’s clinical criteria [26]. According to recently proposed criteria [27,28], 15 MCI patients presented only memory impairment (amnesticMCI), whereas 33 showed impairment not only in memory but also in other cognitive domains (multidomain-MCI). The control group consisted of 24 healthy volunteers matched for ethnic background and age (17 women and 7 men, mean

Table 1 Characteristics of subjects

Gender (M:F) Mean age (years) ± S.E.M. Mean age at onset (years) ± S.E.M. Mean disease duration (years) ± S.E.M. Mean MMSE score ± S.E.M. ApoE ␧4 carriers n (%) *

P = 0.03, AD vs. controls. OR (95%CI): 3.55 (1.12–11.19).

Controls (n = 24)

MCI (n = 48)

AD (n = 94)

7:17 71.5 ± 2.00

19:29 74.7 ± 0.98 73.3 ± 0.90 2.1 ± 0.20 26.7 ± 0.30 15 (33.3)

24:70 79.1 ± 0.72 75.0 ± 0.71 2.6 ± 0.15 18.6 ± 1.23 39 (41.5)*

28.8 ± 1.20 4 (16.6)

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age 71.5 ± 2.0 years). No significant age-related differences were found among groups (P < 0.05). An informed consent to participate in this study was given by all individuals or their caregivers. Both patients and controls were genotyped for the ApoE status. All the information about patients and controls are summarized in Table 1.

using a χ2 goodness of fit test. χ2 was used to test for differences in allele distribution between the groups. The odds ratio (OR) was calculated along with its 95% CI.

2.2. Laboratory methods

Serum MCP-1 levels in both patients and controls are reported in Fig. 1. Significantly increased mean MCP-1 levels were found in MCI as compared with healthy subjects (707.00 ± 33.69 versus 554.81 ± 39.81 pg/ml, P = 0.012, Fig. 1). The same trend was observed considering all AD patients, although the significance level was not reached (664.21 ± 20.93 versus 554.81 ± 39.81 pg/ml, P > 0.05, Fig. 1). However, stratifying AD patients on the basis of the degree of the cognitive impairment in mild to moderate (MMSE > 14) and severe (MMSE ≤ 14) AD, according to the MMSE score at time of sampling, a significant increase of MCP-1 concentration was observed, similarly to MCI, in mild to moderate AD as compared either with severe AD or controls (701.85 ± 24.94 versus 588.73 ± 29.47 or 554.81 ± 39.81 pg/ml, respectively, P < 0.01, Fig. 1). Moreover, these findings were strengthened by a significantly positive correlation between MMSE score and MCP-1 levels in AD patients (ρ = 0.25; P = 0.024, Fig. 2). In addition, despite a small positive effect of age on MCP-1 levels in this group (p = 0.27, P = 0.015, Fig. 3A) a negative

2.2.1. DNA analysis High-molecular weight DNA was isolated from whole blood using a Flexigene Kit (Qiagen, Hildren, Gemany) as described by the manufacturer. ApoE genotype was determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR– RFLP) assay. DNA was amplified using specific primers and then digested with HhaI, as previously described [6]. 2.2.2. MCP-1 determination MCP-1 concentration was measured with a commercial human specific ELISA kit (Amersham), based on the quantitative sandwich enzyme immunoassay technique. The sensitivity of this assay was 10 pg/ml. 2.2.3. Quantitative RT-PCR analysis Total RNA from PBMC of nine patients with AD (six mild and three severe) as well as five MCI and three healthy subjects were extracted with the single step acid phenol method, using Trizol (Invitrogen). RNA was reverse-transcribed using the Ready-To-Go You-Prime First-Strand Beads kit (Amersham Biosciences). For a quantitative estimate of MCP-1 mRNA levels, an ABI 7000 Sequence Detector with duallabeled TaqMan probes was used. The TaqMan MCP-1 probe was employed (Hs00234140ml, Applied Biosystems) and the relative amount of mRNA was determined by comparison with the housekeeping eukaryotic 18S rRNA probe (Hs99999901ml. Applied Biosystems). Final volume reaction was 20 ␮l, using the TaqMan Universal Master Mix (ABI 4324018, Applied Biosystems). Cycle parameters were 2 min at 50 ◦ C and 10 min at 95 ◦ C, followed by 40 cycles of 15 s at 95 ◦ C for denaturation and 1 min at 60 ◦ C for annealing/extension. Relative MCP-1 mRNA levels were calculated as follows: 2−[Ct (sample)−Ct (calibrator)] = 2−Ct , where Ct (sample) [=Ct (MCP-1) − Ct (18S rRNA)] represents the difference, in threshold cycle number, between MCP-1 and 18S rRNA, for a given sample. Ct (calibrator) is the same difference for a healthy control sample to which all samples are compared, as previously reported [35]. 2.2.4. Statistical analysis Statistical analysis was performed using the Sigma Stat 2.0 software. MCP-1 levels were compared using the Mann–Whitney U-test. Spearman test was used for correlations with age or clinical data. ApoE allelic and genotypic frequencies were obtained by direct counting. Hardy Weinberg equilibrium was tested by

3. Results

Fig. 1. Scattergram of MCP-1 levels in serum of MCI, mild/moderate and severe AD patients compared with controls. The black line in each column indicates the mean value. * P = 0.012, MCI vs. CON; ** P < 0.01, mild/moderate AD vs. severe AD or controls.

Fig. 2. Correlation between MMSE score and MCP-1 levels in AD patients (ρ = 0.25). * P = 0.024.

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Fig. 5. MCP-1 basal levels (T0) and after a 1-year follow up (T1) in six MCI patients. Black line: MCI converted to AD; red line: stable MCI; green line: MCI converted to VaD.

Fig. 3. Correlations in AD patients. (A) between age and MCP-1 levels (p = 0.27); (B) between disease duration and MCP-1 levels (ρ = −0.18). * P = 0.015.

correlation between MCP-1 levels and duration of the disease in AD patients was found (ρ = −0.18; P > 0.05, Fig. 3B). A trend toward an increased amount of MCP-1 mRNA was observed, by quantitative real time PCR, in PBMC isolated from a limited group of subjects (five MCI, six mild and three severe AD, as well as three controls). In agreement with levels detected in serum, a trend towards increased MCP-1 mRNA levels in MCI and mild AD as compared with controls was shown (2.6- and 2.2-fold increase over controls, respectively, P > 0.05), whereas in PBMC from severe AD patients the amount of MCP-1 mRNA was similar to controls (Fig. 4). As regards MCI subjects, after a 1-year follow-up, eight of them developed AD, one vascular dementia (VaD), 19 were stable, while the remaining individuals did not present for a control examination anymore. Six subjects (three converted to AD, one to VaD and two stable individuals) gave their consent for a second blood sampling for a further MCP-1

Fig. 4. MCP-1 relative mRNA levels in PBMC from MCI, mild/moderate and severe AD patients compared with controls.

evaluation. All the three subjects converted to AD showed lower MCP-1 levels after conversion than during the MCI phase, although to a different extent (Fig. 5). Conversely, the subject who developed VaD displayed higher MCP-1 levels at the second evaluation, whereas stable MCI had similar levels as compared with the first MCP-1 determination (Fig. 5). As expected [4], the frequency of the ApoE ␧4 allele was significantly higher in AD patients than in controls (41,5 versus 16.6%, P = 0.03, Table 1). The presence of a single copy of this allele increases the risk of developing AD (OR: 3.55, 95% CI: 1.12–11.19). Stratifying patients according to the presence of the ApoE ␧4 allele, no differences in MCP-1 levels were found. Similarly, no differences were observed stratifying by gender.

4. Discussion According to our results, MCP-1 levels are significantly increased in serum from MCI and early AD, but not from severe AD patients, and expression rates in PBMC parallel serum MCP-1 levels. These observations may be further strengthened by the demonstration of a progressive MCP-1 decrease over a 1-year follow up in a subgroup of MCI subjects converted to AD. For the few subjects examined so far, this was true. Although chemokines have traditionally been considered as regulators of inflammatory and immune responses, they also regulate expression of cell death molecules, including caspase-3 [5], Bax and Bcl-2 [2], as well as Fas and its ligand (FasL) [3]. In a mouse model of myocardial ischemia, a pronounced increase in MCP-1 mRNA and protein was detected in the area of injury, suggesting that MCP-1 protects myocytes from hypoxia-induced cell death [34]. Moreover, in MCP-1−/− mice underwent cortical injury, thalamic microglia initially fail to activate, and this delay in microglial activation due to the absence of MCP-1 correlates with a delay in neuronal loss [24]. MCP-1 is involved in the regeneration of neural tissue via the induction of differentiation and a number of neurotrophic factors such as basic fibroblast growth factor (bFGF) by astrocytes and microglia [17].

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Therefore, a possible beneficial role of this molecule would be conceivable. The involvement of cytokines and chemokines in AD is evidenced from several modifications in their concentration in both CSF and serum [36]. Either microglia or other peripheral cells, including monocytes and vascular endothelium, produce and secrete a wide range of cytokines and chemokines, although whether certain biomarkers from the brain are entering the circulation or vice versa is still debated [32]. In our case, five patients with AD were previously included in an analysis of chemokine levels in CSF [9], and the relative samples were collected at the same time of serum samples analyzed in this study. CSF MCP-1 levels in these patients were higher than the correspondent serum levels, and the brain blood barrier (BBB) was intact, as shown by normal IgG indices. Consequently, the observed CSF increased MCP-1 levels were not likely to be related to a disturbance of the BBB function or leakage through it, as also demonstrated by lower serum levels. More recent preliminary findings [10] suggest that CSF MCP-1 levels, despite higher in all AD phases as compared with controls, reach the highest peaks in mild AD, and then decrease in severe AD. These modifications observed in the brain could activate also blood macrophages, which share common functions with microglial cells within the central nervous system (CNS) [31], to produce MCP-1, causing a slight increase in its serum levels, as we describe in this manuscript. Putting together CSF and serum evidences, a possible explanation could be that MCP-1 is increased mostly in CSF during the early phases of AD pathogenesis, but also in serum, to a lesser extent. As the disease progresses, MCP-1 levels decrease progressively either in CSF or in serum, being still significantly detectable only in CSF when comparing with controls. Notably, serum MCP-1 highest peaks have been detected in individuals with MCI, which is considered a transitional state between the cognition of normal ageing and mild dementia [27,28], as well as in AD patients with a mild cognitive decline, suggesting that MCP-1 increase can be restricted to an early stage of the disease. In this regard, previous findings suggest that intrathecal inflammation precedes the development of AD, and represents an initiating factor of the disease rather than a late consequence [9,33]. On the other hand, given the possible neuroprotective effect of MCP-1, its early increase could be related to an attempt of glial cells to remove A␤ plaques, which are the main hallmark of AD, blocking the ongoing neuronal death. As the immunological response fails to restore the normal brain environment, and neurons start dying, cytokine and chemokine production is downregulated, as we observe in late stages of the disease. Despite this process which is supposed to occur in the brain, it is also likely to influence peripheral cells and in accordance with our findings, involve a decreased MCP-1 production with the progression of the disease, as showed by lower MCP-1 levels in three MCI patients who developed AD over a 1-year follow up. The worsening process is probably faster in some individuals than others, as MCP-1 decreases were quite different among these

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three subjects. However, attrition at follow up was so high that findings were not statistically testable. Further confirmation of a generalized decrease of MCP-1 production with the severity of the disease comes from the observed negative correlation between disease duration and MCP-1 levels, despite MCP-1 concentration seems to increase with ageing, as also previously pointed out [15]. However, MCP-1 is probably involved not only in AD pathogenesis, as it is upregulated in different neurodegenerative dementing disorders, such as amyotrophic lateral sclerosis [14,37], suggesting an involvement in a common step linked to neuronal death, possibly as neuroprotective factor. In this regard, the only MCI patient we studied who developed VaD after a 1-year follow up displayed increasing MCP-1 levels over time. Indeed, MCP-1 involvement in VaD is reasonable given the consideration that it looks to play a role in atherosclerosis, acute myocardial infarction and ischaemic stroke [20,30]. In conclusion, these findings confirm that chemokine increase, either in CSF or peripherally, represents a crucial step in pathogenesis and progression of AD. In particular, MCP-1 levels are increased in serum from MCI and mild to moderate AD patients, whereas they decrease during AD progression, suggesting that MCP-1 upregulation is likely to be a very early event in AD pathogenesis, preceding by far the clinical onset of the disease. Nevertheless, as MCP-1 is likely to have a role in several pathologies with an inflammatory component [11], a possible usefulness as an early biomarker to predict conversion from MCI to AD would be possible only in combination with other relevant molecules [23] and after a further independent validation study to assess specificity and sensitivity using ROC analysis. The clinical monitoring of MCI patients who are stable at present is still in progress and will be of help in clarifying these results.

Acknowledgements This work was supported by grants from Associazione “Amici del Centro Dino Ferrari”, CARIPLO and Monzino Foundations, IRCCS Ospedale Maggiore Milano, Centre of Excellence for Neurodegenerative Diseases of the University of Milan, “Associazione per la Ricerca sulle Demenze (ARD)”, and Ing. Cesare Cusan.

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