PLATELET AMYLOID PRECURSOR PROTEIN ISOFORM EXPRESSION IN ALZHEIMER'S DISEASE: EVIDENCE FOR PERIPHERAL MARKER

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1 INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGYAND PHARMACOLOGY Vol. 24, no. 2, (2011) LETTER TO THE EDITOR PLATELET AMYLOID PRECURSOR PROTEIN ISOFORM EXPRESSION IN ALZHEIMER'S DISEASE: EVIDENCE FOR PERIPHERAL MARKER A. VIGNINP, D. SARTINP, S. MORGANTI 1, L. NANETTP, S. LUZZP, L. PROVINCIALP, L. MAZZANTI I and M. EMANUELLP 1Department ofbiochemistry, Biology and Genetics, School ofmedicine, Polytechnic University of Marche, Ancona; 'Department ofneuroscience, Polytechnic University ofmarche, Ancona, Italy Received October 26, AcceptedApril 15, 2011 The first two authors contributed equally to this work Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mrnas prospectively as biomarker for the diagnosis of AD by means of real-time quantitative per, and then evaluated the correlation between APP mrna expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52 fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mrna levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology. Alzheimer's disease (AD), the most common cause of dementia over the age of 65, is a chronic neurodegenerative disorder characterised by a progressive loss of memory and other cognitive functions (1). The search for the molecular determinants of the disease has taken great impulse from the identification of the constituents present in the hallmarks in the brain of AD patients: the amyloid plaques and the neurofibrillary tangles (2). The amyloid plaques are made of a core of insoluble beta-amyloid (A~), a peptide consisting of aminoacids, and several other proteins (3). A~ originates from a larger amyloid precursor protein (APP), by the concerted action ofproteolitic pathways. APP is generated from a large DNA molecule located on chromosome 21. APP gene is alternatively spliced to produce the three major transcripts (APP 770, APP 751 and APP 695) which Key words: Alzheimer, real time-ptlr; mrna, platelets, amyloidprecursorprotein Mailing address: Dr. Arianna Vignini, Department ofbiochemistry, Biology and Genetics School of Medicine, Polytechnic University ofmarche, Via Ranieri 65, 60131, Ancona, Italy Tel: ; Fax: vignini2@libero.it (2011) Copyright by BIOLIFE, s.a.s. This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties

2 530 A. VIGNINI ET AL. contribute to the formation oftotal APP (APP TOT). Once translated, these mrna species generate three protein isoforms constituted of 770, 751 and 695 aa residues. APP 751 and APP 770 contain a Kunitztype serine protease domain (APP KPI), and APP 695 lacks this domain (4-5). Among the different peripheral cells expressing APP forms, platelets are particularly interesting since they show concentrations of its isoforms equivalent to those found in the brain (4). The appropriateness to use platelets, as a cell mirroring some neurochemical processes, finds its rationale in the numerous similar features of platelets and neurones: platelets store and release neurotransmitters, express appropriate neurotransmitter transporters and some neuronerelated proteins such as NMDA receptors (6). Moreover, a number of laboratories independently described alterations in APP metabolism/concentration in platelets ofad patients when compared to control subjects matched for demographic characteristics (7-8). These observations, taken together, identify platelets as an ideal cell for studying pathogenic mechanisms related to Alzheimer's disease associated to the amyloid cascade, and lead to speculate whether platelets could be considered an appropriate cell for studying peripheral diagnostic markers of the disease. We first evaluated the expression of platelet APP mrna isoforms in patients with AD and healthy controls, and then explored the presence ofa correlation between platelet APP mrna expression levels and cognitive measurements. As at present there is no clinical method to accurately identify Alzheimer's disease in the very early phase, the aim of this work is to identify a potential biomarker for the diagnosis ofad. MATERIALS AND METHODS Patients This study was performed on platelets obtained from 18 patients with AD referring to the Neurological Clinic, Polytechnic University of Marche, Ancona (Italy), and 22 healthy control subjects. Inclusion criteria were a diagnosis ofprobable AD according to the NINCDS-ADRDAWork Group under the auspices ofthe Department ofhealth and Human Services Task Force on Alzheimer's Disease (9). All patients and/or their caregivers, and controls gave their written informed consent prior to taking peripheral venous blood samples. The study was performed in accordance with the principles contained in the Declaration ofhelsinki as revised in The study was approved by the local Ethics Committee. Blood samples were collected the first time a diagnosis of Alzheimer's disease was made and no patient was taking medication for AD. Their past medical histories showed no other diseases. General cognitive status was analyzed by using the Mini Mental State Examination (MMSE). Any score greater than or equal to 25 points (out of30) is effectively normal while reduced scores can indicate severe (~9 points), moderate (10-20 points) or mild (21-24 points) cognitive impairment (10). Preparation ofplateletpellets Peripheral venous blood samples were taken after overnight fasting, and were immediately mixed with anticoagulant EDTA. Platelets were isolated by differential centrifugation according to our previous studies (II). The method involved a preliminary centrifugation step at 200xg for 10 min to obtain platelet-rich plasma (PRP). The PRP was centrifuged at 2000xg for 20 min to isolate the platelets and immediately stored at -80 C until use. RNA isolation and edna synthesis Total RNA was isolated from whole platelet pellets using RNeasy Micro Kit (Qiagen), according to the manufacturer's protocol. RNA samples were reverse transcribed in a total volume of20 ul for 120 min at 37 C with High Capacity edna Reverse Transcription Kit (Applied Biosystem), using random primers. Real-time quantitative per To examine expression of platelet-derived APP isofonns we performed real-time PCR assay using the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad Laboratories). cdnas generated as previously described, were used for real-time quantitative PCR. To avoid false-positive results attributable to amplifying contaminating genomic DNA in the edna preparation, all primers were selected to flank an intron. Also, PCR efficiency of each primer pair was tested (Table II) and found to be close to 1. Genes were run in duplicate for 40 cycles at 94 C for 30 sec and 56 C for 30 sec using the iq SYBR Green Supennix (Bio-Rad Laboratories). All samples were tested in triplicate with the reference gene ~-actin for data normalization to correct for variations in RNA quality and quantity. Direct detection of PCR products was monitored by measuring the fluorescence produced by SYBR Green I dye binding to double strand DNA after every cycle. These measurements were then plotted against cycle numbers. The parameter threshold cycle (Ct) was defined as the cycle number at which the first

3 Int. J. ImmunopatboI. Pbarmacol. 531 detectable increase above the threshold in fluorescence was observed. Fold changes in relative gene expression were calculated using the equation, 2- Me, where dct = Ct (APP) - Ct (~-actin) and ddct = mean-dct (AD patients) - mean-dct (controls). In addition, dct values were used to compare the expression levels of platelet APP mrnas with cognitive status (MMSE) of examined subjects. A small dct value indicates a high expression level, while a large dct value is attributable to a low expression level. Statistical analysis Data were analyzed using GraphPad Prism software version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). Mean-dCt was calculated as the average (± standard deviation) of all dct values within each group ofsamples (AD and control samples). Differences in gene expression levels between the AD group and the control group were determined using the Mann-Whitney test. Differences in age and MMSE scores, between patients and controls, were evaluated by Student's t-test, while differences in gender by Fisher exact test. Pearson's correlation coefficient (p) was used to test relations between platelet APP mrna levels.(dct values) and cognitive measurements (MMSE scores). Differences were considered significant at p < RESULTS Clinicopathologicalfindings The clinical and demographic characteristics of the recruited subjects are summarized in Table I. Groups were matched for age (p=ns) and sex (p=ns). Since coincident sampling was used, there were no significant differences between patients and controls with regard to time or date of blood sampling. All patients with AD had an MMSE performance significantly lower than normal subjects (l7.4±3.7 vs 28.9±1.8 respectively; p<o.ooi). On the basis of the score that the AD patients reached on the MMSE they were classified in the mild-moderate stage ofad. Analysis ofplatelet APP isoforms expression with quantitative real-time PCR Real-time quantitative PCR was used to evaluate the expression of APP isoforms in platelets of 18 patients with AD and 22 controls. Differential gene expression measurements (AD versus control) revealed in the AD patient group a significant upregulation ofapp TOT (1.52-fold), APP KPI (1.32 fold), APP 770 (1.33-fold) andapp 751 (1.26-fold). No significant difference was detected for APP 695 (Table III). Correlations between APP mrnas levels arid cognitive measurements When all the enrolled subjects were considered (n=40), a statistically significant positive correlation was found between APP mrnas levels (TOT, KPI, 770 and 751) and cognitive impairment. APP 695 mrna expression levels showed no significant relationship with MMSE scores (Table IV). DISCUSSION Real time-pcr experiments demonstrated that all APP isoforms from AD platelets were upregulated compared to control platelets except the APP695 isoform. Previous studies demonstrated that Alzheimer patients show concentrations of APP isoforms (APP770, APP751 andapp695) in platelets equivalent to those found in the brain (4). These observations indicate that the blood platelet APP is processed by the same amyloidogenic and nonamyloidogenic pathways as utilized in the brain. Platelets can be considered asa source of human biological material available for the study of the metabolic mechanisms mirroring, in the peripheral Table I. Main characteristics ofcontrols and Alzheimer disease patients (AD)..' Controls (n = 22) AD (n =18) p value Age range 76±6 79±4 NS Sex (MIF) 10/12 7/11 NS MMSE 28.9± ±3.7 < Results are expressed as Means±S.D.

4 532 A. VIGNINI ET AL. Table II. Gene specific primers usedfor quantitative Real- Time PCR. TargetmRNA Sequence Product length (bp) APPTOT Forward 5' -aaccagtgaccatccagaac-3' 129 Reverse 5' -acttgtcaggaacgagaagg-3' AP.P KPI Forward 5'-gtctgtggaagaggtggttc-3' 154 Reverse 5'-gtcaaagttgttccggttg-3' APP770 Forward 5' -cccgagatcctgttaaacttc-3' 119 Reverse 5'-cctctctttggctttctgg-3' APP 751 Forward 5'-cagcgccattcctacaacag-3' 109 Reverse 5' -cctctctttggctttctgga-3' APP695 Forward 5'-ggtggttcgagttcctacaa-3' 112 Reverse 5'-cctctctttggctttctgga-3' ~-actin Forward 5'-ctcttccagccttccttcct-3' 116 Reverse 5'-agcactgtgttggcgtacag-3' Table III. Expression levels ofappplatelet isoforms in AD patients comparedwith controls. Target ACt in AD ACt in controls AD/control mrna patients (n = 18) (n = 22) (-foldt p-value" APPTOT 6.26± ± APPKPI 7.06± ± APP ± ± APP ± ± APP ± ± !i.Ct values are mean ± standard deviation. "Differential gene expression measurements were performed by Real-Time PCR, as described in Materials andmethods. "Statistical significance was set at p < Table IV. Correlations between!i.ct values and MMSE scores in all enrolled subjects. APPTOTACt APP KPI ACt APP770ACt APP751 ACt APP695 ACt MMSE score p=o.5l3 p=o.350 p=o.377 p=o.364 p=-o.20l (n=40) p=o.ooi p=o.034 p=o.022 p=o.029 p=o.336

5 Int. J. Immunopathol. Pharmacol. 533 compartment, the evolution of the biochemical processes occurring in the central nervous system and related to Alzheimer's disease. Cattabeni et al., (4) evaluated the concentrations of APP forms in whole platelet homogenate by means of Western blot analysis and found an altered APP ratio in platelets obtained from patients affected by AD. This altered ratio showed a positive correlation with the severity ofthe disease. However, the authors were not able to detect differences in APP mrna levels between the two groups, because of the use of a semiquantitative procedure, and they ascribed the observed decrease to a post-translational modification ofapp protein. By contrast, the sensitivity and specificity of Real-time PCR procedure allowed us to quantify APP isoform mrna levels and show that higher levels of platelet-derived APP isoforms are present in platelets with AD. All splice variants, once translated, can contribute to the synthesis of the full length APP, which undergoes post-translational modifications. Classical N-glycosylation and O-glycosylation occur during transit through the endoplasmic reticulum (ER) and the Golgi apparatus. Addition of sulphate and phosphate in the late Golgi compartment further increases the structural complexity of APP. Moreover, APP is then proteolytically cleaved by a-, B- and y-secretase (12), releasing different fragments with different molecular weights. Borroni et al. (13) evaluated the levels ofapp forms by Western blot analysis with monoclonal antibody 22Cll raised against the N-terminal domain of APP, therefore recognizing the three APP forms with apparent molecular weight of 130, 110 and 106 kda. In this light, it is hard to find a significant correlation linking the reduced ratio between the upper (130 kda) and the lower (106 1iO kda) immunoreactive bands and our results, regarding mrna expression levels. The present results are consistent with the hypothesis that platelet APP could be considered a potential peripheral marker for studying AD. In particular, the role of platelet APP mrnas as biomarker could contribute to define a signature for the presence ofad pathology. The significant positive correlation between APP transcript levels and the severity of the cognitive impairment suggests that one interesting future goal might be the long-term follow-up of the ADNI (Alzheimer's Disease Neuroimaging Initiative) cognitively normal individuals, in order to speculate whether the evaluation ofapp transcript levels could be used for an accurate prediction ofad pathology. ACKNOWLEDGEMENTS The present work was supported by an RSA grant from the Polytechnic University ofmarche to LM. REFERENCES 1. Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med 2010; 362: Blennow K, Hampel H, Weiner M, Zetterberg H. Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol 2010; 6: Zhang YW, Thompson R, Zhang H, Xu H. APP processing in Alzheimer's disease. Mol Brain 2011; 4:3. 4. Cattabeni F, Colciaghi F, Di Luca M. Platelets provide human tissue to unravel pathogenic mechanisms of Alzheimer disease. Prog Neuropsychopharmacol BioI Psychiatry 2004; 28: Matsui T, Ingelsson M, Fukumoto H, Ramasamy K, Kowa H, Frosch MP, Irizarry MC, Hyman BT. Expression ofapp pathway mrnas and proteins in Alzheimer's disease. Brain Res 2007; 1161: Di Luca M, Colciaghi F, Pastorino L, Borroni B, Padovani A, Cattabeni F. Platelets as a peripheral district where to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein. Eur J Pharmacol2000; 405: Di Luca M, Pastorino L, Bianchetti A, Perez J, Vignolo LA, Lenzi GL, Trabucchi M, Cattabeni F, Padovani A. Differential level of platelet amyloid beta precursor protein isoforms: an early marker for Alzheimer disease. Arch Neurol 1998; 55: Baskin F, Rosenberg RN, Fang X, Hynan LS, Moore CB, Weiner M, Vega GL. Correlation of statinincreased platelet APP ratios and reduced blood lipids in AD patients. Neurology 2003; 60: McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of

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