Osteopontin is Increased in the Cerebrospinal Fluid of ... - IOS Press

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Journal of Alzheimer’s Disease 19 (2010) 1143–1148 DOI 10.3233/JAD-2010-1309 IOS Press

Short Communication

Osteopontin is Increased in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease and Its Levels Correlate with Cognitive Decline Cristoforo Comia,b,c,∗ , Miryam Carecchiob , Annalisa Chiocchettia,d , Stefania Nicolaa,d , Daniela Galimbertie , Chiara Fenoglioe, Giuseppe Cappellano a,d, Francesco Monaco a,b, Elio Scarpinie and Umberto Dianzani a,d a

Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), “A. Avogadro” University of Eastern Piedmont, Novara, Italy b Department of Neurology, “A. Avogadro” University of Eastern Piedmont, Maggiore Hospital, Novara, Italy c Neurorehabilitation Institute “M.L. Novarese”, Moncrivello (VC), Italy d Department of Medical Sciences, “A. Avogadro” University of Eastern Piedmont, Maggiore Hospital, Novara, Italy e Department of Neurological Sciences, Dino Ferrari Center, University of Milan, IRCCS Ospedale Maggiore Policlinico, Milan, Italy

Accepted 22 October 2009

Abstract. Inflammation is believed to play a role in Alzheimer’s disease (AD). Osteopontin (OPN) is a molecule involved in macrophage recruitment and activation and implicated in neurodegeneration. In order to elucidate the role of OPN in AD, we evaluated its levels in serum and cerebrospinal fluid (CSF) of 67 AD patients, 46 frontotemporal dementia (FTD) patients, and 69 controls. We found that OPN levels: i) are significantly increased in the CSF of AD patients; ii) correlate with MMSE score; and iii) are higher in the early disease phases ( 2 years). These findings support a role of OPN in AD pathogenesis. Keywords: Disease progression, early Alzheimer’s disease, Eta-1, microglia, neuroinflammation

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment. Its two major neuropathologic hallmarks are ∗ Correspondence

to: Cristoforo Comi, MD, PhD, Department of Neurology, University “A. Avogadro” of Eastern Piedmont, via Solaroli 17, I-28100 Novara, Italy. Tel.: +39 0321 3733965; Fax: +39 0321 3733298; E-mail: [email protected].

extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles, composed of the tau protein. In AD brains, deposition of Aβ is closely associated with an inflammatory response with local upregulation of acute-phase proteins, cytokines, and other inflammatory mediators [1]. Under inflammatory conditions, microglia cells, which are the resident macrophages of the brain, are activated and further support inflammation contributing to neuronal damage and disease progression [2].

ISSN 1387-2877/10/$27.50  2010 – IOS Press and the authors. All rights reserved

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C. Comi et al. / Osteopontin in AD

Osteopontin (OPN) is a phosphoprotein highly expressed by macrophages. Substantial evidence supports a proinflammatory role of OPN, acting as a cytokine in regulating macrophage function in response to inflammatory stimuli [3]. Macrophage migration and pro-inflammatory cytokine production are impaired in the absence of OPN [4]. OPN expression is increased in pyramidal neurons of AD patients and in the brains of AβPP/PS1KI mice, an experimental model of AD [5,6]. Intriguingly in AD brains, the increase in OPN expression is closely associated with Aβ deposition [5]. Given these premises, the aim of this work was to further elucidate the role of OPN in AD: 1) by assessing its serum and cerebrospinal fluid (CSF) levels in AD compared to frontotemporal dementia (FTD) patients and controls and 2) by comparing OPN CSF levels in patients with different degrees of cognitive decline.

MATERIALS AND METHODS Patients 67 patients with AD [7], 46 patients with FTD [8], and 69 controls were enrolled in the study (Table 1). Patients underwent a standard evaluation, including medical history, physical and neurological examination, screening laboratory tests, neuropsychological evaluation, brain MRI or CT and, if indicated, PET. Significant vascular brain damage was excluded (Hachinski Ischemic Score < 4). In the majority of cases, the severity of dementia at diagnosis was assessed by the Mini Mental State Examination (MMSE) [9] (48/58 of AD and 40/46 of FTD patients), and disease duration was determined as the time in years between the first symptoms and diagnosis (50/67 AD and 36/46 FTD patients). 23 AD patients were diagnosed at a very mild stage (MMSE 24–27) thanks to new research criteria [7]. For each patient, serum and CSF were collected at diagnosis. Patients or caregivers gave informed consent to lumbar puncture and to CSF and serum analyses for both diagnostic and research purposes. An aliquot of CSF was used to evaluate Aβ, tau, and P-tau levels that provide a more accurate diagnosis [7], and a second aliquot was used to measure OPN levels. After recruitment, a follow-up of patients was performed and the initial diagnosis of either AD or FTD was confirmed in all subjects. The control group was composed by 69 subjects matched for ethnic background, age, and gender (Ta-

ble 1), with acute headache, intracranial hypotension, compressive radiculopathy, or non-immune peripheral neuropathy who underwent lumbar puncture during their diagnostic workup. All controls had normal CSF findings, no memory complaints, and no signs of inflammatory, neoplastic, or neurodegenerative disorders. They gave informed consent to participate in the study. The study was approved by the local ethical committee. All the experiments were performed in accord with the Helsinki Declaration of 1975. CSF/serum sample collection and routine analysis CSF samples were obtained by lumbar puncture at the L4/L5 or L3/L4 interspace, centrifuged at 4 ◦ C and stored at −30◦C until analysis. CSF cell counts, glucose, and proteins were determined. Albumin was measured by rate nephelometry. To evaluate the integrity of the brain blood barrier and the intrathecal IgG production, the albumin quotient (CSF albumin/serum albumin) x 10 3 and the IgG index (CSF albumin/serum albumin)/(CSF IgG/serum IgG) were calculated [10]. ELISA assays Serum and CSF OPN concentrations were evaluated in a capture enzyme-linked immunoadsorbent assay (ELISA) according to the protocol provided by the manufacturer (Calbiochem, San Diego, CA). All assays were performed in duplicate, and the observer (S.N.) was blinded to the diagnosis. The optical density was measured with a microplate reader (Bio-Rad, Hercules, CA). The I-smart program was used to create a regression curve. Aβ 42 , tau, and P-tau CSF levels were evaluated with specific ELISA kits (Innogenetics) [11]. Statistical analysis For the ELISA data, the approximation of population distribution to normality was tested. Results were asymmetrically distributed and consequently presented as median values and interquartile ranges. ELISA data comparisons were performed with the Mann-Whitney U-test. Correlations between OPN levels and clinical parameters were tested with the Spearmann Correlation Test.


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Table 1 Clinical features and laboratory findings of healthy controls and AD and FTD patients Gender (M:F) Age at samplinga (mean ± SD) Age at onset (mean ± SD) Disease durationb (mean ± SD) MMSE (median value, interquartile range) OPN CSF levels, (ng/ml) (median value, interquartile range) OPN serum levels (ng/ml) (median value, interquartile range) Aβ CSF levels (pg/ml) (median value, interquartile range) Tau CSF levels (pg/ml) (median value, interquartile range) P-Tau CSF levels (pg/ml) (median value, interquartile range)

Controls 29:40 67 ± 15

29 (28-30) 2562 (1355–4680) 150 (72–220) 995 (708–1204) 160 (104–223) 28 (20–34)

AD 28:39 70 ± 12 62 ± 14 4±2 22 (21-25) 5968 (2664–10762)c 165 (89–258) 375 (321–480) 640 (491–898) 57 (52–84)

FTD 19:27 69 ± 8 64 ± 9 3±2 22 (20-24) 3239 (2000–6281) 170 (86–295) 812 (426–1112) 441(281–738) 41 (28–69)

a CSF

collected at time of diagnosis. in years between the first symptoms (by history) and the clinical diagnosis. c p = 0.02 AD versus FTD and < 0.0001 AD versus controls. b time

RESULTS First, we compared OPN levels in serum and CSF of AD patients, FTD patients, and controls. We found that OPN levels in the CSF were significantly higher in AD patients than in both FTD patients and controls (p = 0.02 and < 0.0001 respectively), whereas no difference was found between FTD patients and controls (p = 0.2). Regarding the serum levels of OPN, we found no difference among the groups (Table 1). Secondly, we explored whether there was a correlation between the levels of OPN in the CSF of AD patients and both the degree of cognitive decline and disease duration. We found that OPN CSF levels displayed direct correlation with the MMSE score (r = 0.58, p < 0.0001, Fig. 1A) and inverse correlation with disease duration (r = − 0.40, p = 0.003, Fig. 1B). Thereafter, we stratified patients according to their MMSE score and disease duration. We found that patients with MMSE score > 23 (n = 23) displayed significantly higher OPN CSF levels than patients with MMSE 23 (n = 34) (median values 10582 versus 3732 ng/ml; interquartile ranges 12326-5211 versus 7101-1625; p = 0.0004 Fig. 1C). Moreover, patients with a disease duration 2 years (n = 21) displayed significantly higher levels than patients with disease duration > 2 years (n = 29) (median value 10942 versus 4142 ng/ml; interquartile ranges 12319-6361 versus 7129-1968; p = 0.003 Fig. 1D). On the contrary, no significant correlation was found between OPN CSF levels and gender, age, and Aβ42 , tau, or P-tau levels (data not shown). DISCUSSION This work shows that AD patients display about a two-fold increased OPN levels in the CSF compared to

age-matched controls. Moreover, this finding is specific for AD, since it is not present in FTD patients and is particularly striking in the early stages of the disease. In previous reports, OPN had been detected in brains of AD patients by immunocytochemistry [5], and an increase of an OPN fragment was detected by SELDITOF-mass spectrometry in the CSF of subjects with mild cognitive impairment who progressed to AD [12]. Our findings suggest that OPN may be involved in AD pathogenesis and fit with the role ascribed to inflammation in the early phases of the disease. This role has been suggested by previous reports detecting an increase of inflammatory molecules, such as IP-10 and IL-11, in the CSF of patients in the initial stages of AD [13,14]. Most researchers believe that this inflammatory response is detrimental for the central nervous system environment and that pro-inflammatory cytokines, such as TNF-α and IFN-γ further support this inflammation in a loop fastening neurodegeneration [1]. On the contrary, it has been previously suggested that inflammation may be a beneficial response reacting against the tissue degeneration and possibly stimulating regenerative processes [15]. The high level of OPN might have two opposite roles as well. On the one hand, data from Simonsen and colleagues suggest that OPN may favor AD development, since MCI patients who have high CSF levels of an OPN fragment are more likely to progress to AD [12]. This possibility is supported by studies on the role of OPN in other neurodegenerative diseases. Most reports involve multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) and point to a detrimental role of OPN. MS patients display high levels of OPN in the brain, CSF and serum; these levels increase during MS relapse and variants of the OPN gene causing secretion of high OPN levels seem to favor fast disease progression [16]. In EAE, OPN

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A

B

C

D

Fig. 1. OPN CSF levels in AD patients. A) Correlation between OPN CSF levels and MMSE score in AD patients (r = 0.58, p < 0.0001). B) Correlation between OPN CSF levels and disease duration in AD patients (r = − 0.4, p = 0.003). C) Median OPN CSF levels in patients stratified according to the MMSE score. Vertical bars show interquartile ranges (p = 0.0002). D) Median OPN CSF levels in patients stratified according to disease duration. Vertical bars show interquartile ranges (p = 0.003).


knockout mice are resistant to disease progression [17] and display disease exacerbation after injection of recombinant OPN [18]. A detrimental role has also been suggested in Parkinson’s disease (PD) since PD patients display increased OPN levels in serum and CSF, particularly those with dementia [19]. Moreover, OPN knockout mice display lower nigral cell death and glial response than wild-type mice, following 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment [19]. On the other hand, it was suggested that OPN may also have neuroprotective/regenerative functions. Iczkiewicz and colleagues postulated that the decrease of OPN expression found in residual nigral dopaminergic neurons in PD and in the related degenerative disorders, multiple system atrophy and progressive supranuclear palsy, indicated that this molecule might have a neuroprotective role [20]. On the regenerative side, it was shown that OPN administration may enhance myelin formation in vitro [21]. Our data do not bring light into this questioning, but some light might derive from future follow-up of newly diagnosed AD patients assessing whether high OPN levels in the CSF in the early disease phases may be a prognostic factor of diseases progression.

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ACKNOWLEDGMENTS This work was supported by Compagnia di San Paolo (Turin), Regione Piemonte “Ricerca Finalizzata” project and “Ricerca Applicata CIPE” project (Turin), FISM grant 2008/R/11 (Genoa), AIRC (Milan). Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=186). REFERENCES [1] [2]

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