Cholinesterase inhibitors influence APP metabolism in Alzheimer disease patients

www.elsevier.com/locate/ynbdi Neurobiology of Disease 19 (2005) 237 – 242

Cholinesterase inhibitors influence APP metabolism in Alzheimer disease patients Martina Zimmermann,a,T Barbara Borroni,b Flaminio Cattabeni,a Alessandro Padovani,b,1 and Monica Di Lucaa,1 a

Centre of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy Department of Medical Sciences-Neurology Unit, University of Brescia, Piazza Spedali 1, 25125 Brescia, Italy

b

Received 29 October 2004; revised 14 December 2004; accepted 4 January 2005 Available online 17 February 2005

Platelets mirror pathogenic alterations in the central nervous system of Alzheimer disease (AD) patients: an alteration of the Amyloid Precursor Protein (APP) forms pattern and decreased alphasecretase activity – the non-amyloidogenic APP processing enzyme – were demonstrated. Platelets were analysed at baseline and after 30 days of cholinesterase inhibitor (ChEI) treatment (T30). ADAM10 levels, alpha- and beta-secretase activity were assessed measuring ADAM10 immunoreactivity, sAPPalpha release and the membrane-attached C-terminal fragments produced by beta- and alpha-secretase cleavage, that is, CTF99 and CTF83, respectively. ChEIs treatment rescues impaired APP metabolism increasing significantly ADAM10 levels (T30 vs. T0, P b 0.05), alpha-secretase activity (T30 vs. T0, P b 0.05) and reducing beta-secretase cleavage (T30 vs. T0, P b 0.05). Restoration of the balance between the mutually exclusive alpha- and beta-secretase pathway in APP processing caused by short-term ChEIs treatment potentially represents a key event in AD therapy linking in vivo cholinergic effect to APP metabolism. The use of platelets may represent a useful tool to follow molecular aspects of pharmacological response in AD patients. D 2005 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Platelets; APP metabolism; Cholinesterase inhibitors; Alpha-secretase; Beta-secretase; ADAM10

Introduction Pharmacological treatment of Alzheimer disease (AD) still represents an unsolved issue. Significant improvements in cognition and global function have been observed upon treatment with ChEIs (Doody, 1999; Farlow, 2002; Rogers and

T Corresponding author. Fax: +39 02 50318284. E-mail address: [email protected] (M. Zimmermann). 1 AP and MdL contributed equally to direction and design of this study. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.01.002

Friedhoff, 1996), though clinical research has only been able to verify the symptomatic effect of these treatments (Eagger and Harvey, 1995; Farlow, 2002). In clinical studies, however, a considerable number of AD patients show a long-lasting cognitive stabilisation after ChEIs treatment, which cannot be solely explained by a pure symptomatic effect of this class of drugs but rather it suggests a disease modifying action (Giacobini, 2001, 2003). Accordingly, in vitro studies claim for ChEIs an effect on the amyloid precursor protein (APP) metabolism (Racchi et al., 1999), a biological pathway causally related to AD pathogenesis (Sinha and Lieberburg, 1999). APP that is present in cells in different isoforms can, in fact, be cleaved by either alpha- or beta-secretase, whereby the former prevents amyloid formation, releases a soluble N-terminal APP fragment sAPPalpha and leaves a C-terminal fragment (CTF) attached to the cellular membrane (CTF83), while the latter is instrumental for production of CTF99 and amyloid beta (Nunan and Small, 2000). To test this hypothesis in vivo, a reliable biomarker which may help us in tracing the effect of ChEIs on APP metabolism, is mandatory. We have previously reported that both the ratio of APP forms and the levels of ADAM10 (A Disintegrin And Metalloprotease which is the most accredited candidate for alphasecretase; Lammich et al., 1999) are significantly reduced in AD patients’ platelets when compared to controls, thus being considered as accurate biomarkers of the disease (Baskin et al., 2000; Colciaghi et al., 2004; Di Luca et al., 1998; Padovani et al., 2001; Rosenberg et al., 1997). The aim of this study is, therefore, to determine whether short-term ChEIs treatment affects APP metabolism in vivo. To this end, the major APP metabolites, that is, sAPPalpha and the membrane-attached CTF83 and CTF99 produced by alpha- and beta-secretase cleavage, respectively, were measured in platelets of treated AD patients. A time interval of 30 days of donepezil treatment was chosen, since this time range proved itself useful in rescuing APP forms in platelets (Borroni et al., 2001). Results of these experiments will provide proof at a molecular

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level for the in vivo efficacy of ChEIs in the treatment of AD pathology.

secondary antibodies (Kirkegard, Gaithersburg, MD, USA; dilution 1:10,000) for 45 min, blots were developed with enhanced chemiluminescence (Amersham-Pharmacia Biotech, Little Chalfont, UK).

Materials and methods

Platelet preparation

Characteristics of the subjects

Personnel carrying out platelet preparation and subsequent analysis was blind for diagnosis and treatment. Platelets were obtained at baseline and after 30 days of ChEIs treatment. 27 ml of blood was collected into 1 vol 3.8% sodium citrate (in the presence of 136 mM glucose), mixed gently and centrifuged at 200  g for 10 min. The time interval between blood drawing and the first centrifugation was never longer than 20–25 min. Platelet rich plasma was separated from blood cells using a plastic pipette, carefully avoiding the drawing in of the buffy coat. Platelets were collected by centrifugation at 500  g for 20 min. Platelet pellets were washed twice with Tris–HCl 10 mM pH 7.4 and resuspended in an ice-cold lysis buffer (L-buffer) containing Tris–HCl 10 mM pH 7.4, EGTA 1 mM, PhenylMethyl-Sulfonyl Fluoride 0.1 mM, and a complete set of protease inhibitors (Complete, Roche, Mannheim, Germany). Homogenates were then subjected to 3 rounds of freeze thawing followed by 15 s of sonication at 08C.

The study was undertaken on 25 AD patients and on 25 ageand gender-matched control subjects (CON) (Table 1). The diagnosis of probable AD was made according to the National Institute of Neurological Disorders and Stroke-Alzheimer Disease and Related Disorders Association (NINCS-ADRDA) criteria. Exclusion criteria for all groups were the following: head trauma, metabolic dysfunctions, haematological diseases, alcohol abuse, drug abuse, delirium, mood disorders, treatment with medications affecting platelet functions, that is, anticoagulants, antiplatelet drugs, serotoninergic agonists–antagonists, and corticosteroids. All subjects included had a standardised clinical workup based on neurological examinations, laboratory blood and urine analysis, neuropsychological assessment including a minimental state examination (MMSE) and a clinical dementia rating (CDR), and neuroimaging study (Head Computed Tomography and/or Magnetic Resonance Imaging). Before enrolment, subjects or their legal caregivers filled out an informed consent, after the nature and possible consequences of the study was explained. Study design: this was a 4-week, longitudinal study. At baseline, AD patients and control subjects were subjected to the abovementioned clinical evaluation, including a venipuncture for platelet sample collection. AD patients were treated with donepezil, 5 mg/ day, receiving a single dose of donepezil each evening. After 30 days, AD patients underwent a further venipuncture for platelet collection. Antibodies Immunostaining reactions were performed incubating with the monoclonal antibody (mAb) 22C11 (Chemicon, Temecula, CA, USA; dilution 1:3,000) for total APP, mAb 6E10 (Chemicon, Temecula, CA, USA; dilution 1:3,000) for sAPPalpha, polyclonal antibody (pAb) anti-ADAM10 (ProSci Inc., CA, USA; dilution 1:1,000) for ADAM10 and pAb anti-Actin (Sigma-Aldrich, St. Louis, MO, USA; dilution 1:3,000) for actin in 3% non-fat milk, and mAb 4G8 (Chemicon, Temecula, CA, USA; dilution 1:1,000) for CTFs in albumin 5%. After incubation with peroxidase conjugated Table 1 Demographic and clinical characteristics of AD patients and control subjects Variables

AD

CON

P

N Age, years Gender, F/M Education, years Estimated disease duration, years MMSE baseline CDR baseline

25 75.0 F 5.0 18/7 6.18 F 2.97 2.13 F 1.14

25 73.5 F 5.6 15/10 6.2 F 2.65 –

– n.s. n.s. n.s. –

21.6 F 5.7 0.98 F 0.67

29.2 F 0.8 0.00

0.0001 0.0001

AD: Alzheimer Disease patients; CON: Control subjects; F: Female; M: Male; MMSE: Mini-Mental State Examination; CDR: Clinical Dementia Rating scale.

Platelet activation Platelet pellets obtained from 2 ml of platelet rich plasma were washed and resuspended in Tyrode’s buffer according to Bush et al. (1990) and incubated with thrombin (1 U/ml) (Sigma-Aldrich, St. Louis, MO, USA) in the presence of MgCl2 and CaCl2 (T, activation condition) and in the absence of both MgCl2 and CaCl2 (B, basal condition), stirring for 15 min at room temperature. After activation, platelets were pelleted by centrifugation at 800  g for 15 min and resuspended in L-buffer. The supernatants were desalted using DG10 columns (Biorad-Lab., CA, USA), lyophilised and resuspended in Laemmli buffer and separated on 6% SDS-PAGE and electroblotted onto nitrocellulose membranes. sAPPalpha staining and quantification was carried out as described. CTF measurements Platelet proteins were separated in 13% Tris-Tricine SDSPAGE and electroblotted. Immunostaining reactions were performed incubating overnight using mAb 4G8, which is directed against the sequence of amino acids 17–21 of the Abeta domain, hence, detecting in the same sample and the same blot both the alpha- and beta-secretase cleaved product CTF83 and CTF99. After incubation with peroxidase conjugated secondary antibodies blots were developed with enhanced chemiluminescence (Femto, Pierce, Rockford, IL, USA). sAPPalpha measurements sAPPalpha released from activated platelets was quantified as previously described (Colciaghi et al., 2002). Briefly, two platelet pellets obtained from the same patient were activated under B and T conditions in parallel; both platelet pellets were obtained from the same amount of plasma rich in platelets (2 ml). The actual release of sAPPalpha was then calculated as the immunoreactivity of the protein bands found under T conditions versus the immunoreactivity of the protein bands detected under B conditions.

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ADAM10 quantification Western blot (WB) experiments were performed as previously described (Colciaghi et al., 2004) and actin was used as internal control and standard. Data analysis and statistical evaluation Quantification of WB analysis of ADAM10 and CTFs in platelets and sAPPalpha in supernatants was performed by means of computer assisted imaging (Quantity-OneR System, Biorad, CA, USA). For the analysis of ADAM10 levels, the ratio between the immunoreactivity of the ADAM10 band at 68 kDa and the immunoreactivity of the actin band at 48 kDa was calculated. For the CTF ratio, the ratio between the immunoreactivity of the CTF99 band and the immunoreactivity of the CTF83 band was calculated. For the analysis of sAPPalpha release, see above. Data are expressed as mean F SEM, results are obtained from 25 AD patients and 25 CON subjects and individual data replicated three times for intra-individual variations. Statistical evaluations were performed according to a one-way analysis of variance followed by Bonferroni as a post hoc comparison test.

Results Characterisation of the secretases’ system in vivo in na¨lve AD patients Firstly, the levels of ADAM10 in platelets of AD patients were measured by WB analysis and compared to ADAM10 levels in CON subjects. Representative ADAM10 immunoreactivity from CON and AD is shown in Fig. 1A. The upper and lower protein bands correspond to ADAM10 and actin, respectively. The ratio between the immunoreactivity of ADAM10 versus actin was measured: a marked decrease of the ADAM10/actin ratio was observed in AD patients when compared to CON (Fig. 1A; mean F SEM CON: 0.82 F 0.06, AD: 0.39 F 0.05; P b 0.05).

Fig. 1. The secretases’ system in platelets of control subjects and naRve AD patients. (A) ADAM10 and actin in platelets of control subjects and AD patients. Top: two representative WB analyses of the metabolically active form of ADAM10 (open arrowhead, 68 kDa apparent MW) and the internal standard actin (black arrowhead, 48 kDa apparent MW) in two control subjects (CON 1, CON 2) and two AD patients (AD 1, AD 2) are shown. Bottom: ADAM10/actin ratio is significantly decreased in AD vs. CON ( P b 0.05). (B) Release of sAPPalpha from activated platelets in control subjects and AD patients. Top: two representative WBs of sAPPalpha release from thrombin activated platelets in two control subjects (CON 1, CON 2) and in two AD patients (AD 1, AD 2). Bottom: quantitative analysis of the release of sAPPalpha from activated platelets in control subjects and AD patients, measured as ratio of thrombin provoked release versus basal release T/B. The release of sAPPalpha from thrombin activated platelets of AD patients is significantly lower when compared to controls (CON) ( P b 0.05). (C) CTF99 and CTF83 in platelets of control subjects and AD patients. Top: two representative WB analyses of CTF99 and CTF83 (12 and 10 kDa apparent MW, respectively) in two control subjects (CON 1, CON 2) and two AD patients (AD 1, AD 2). Bottom left: quantitative analysis: the immunoreactivity of the CTF99 in AD patients is significantly higher than that found in control subjects (CON) ( P b 0.05). Bottom right: the CTF99/CTF83 ratio is significantly increased in AD patients when compared to control subjects ( P b 0.01).

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Concomitantly, sAPPalpha released from activated platelets of CON subjects and AD patients was measured, as an index of alphasecretase activity. As shown in Fig. 1B sAPPalpha released from platelets of AD patients was significantly lower than that observed from CON (Fig. 1B; mean F SEM CON: 1.5 F 0.2, AD: 1.1 F 0.2; P b 0.05). Further, CTF83 and CTF99, generated by alphaand beta-secretase activity, respectively, were analysed and quantified. Fig. 1C shows representative WBs of CTF99 and

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CTF83 in CON subjects and AD patients. The immunoreactivity of the CTF99 band was significantly higher in AD patients when compared to CON (Fig. 1C bottom left; mean F SEM CON: 96.0 F 10.6, AD: 155.7 F 19.4; P b 0.05). The immunoreactivity of CTF83 showed a trend of decrease in AD patients even though it did not reach a statistical criterion. The calculated ratio between the immunoreactivity of CTF99 (12 kDa) and of CTF83 (10 kDa) was significantly increased in AD patients when compared to CON (Fig. 1C bottom right; mean F SEM CON: 0.30 F 0.04, AD: 0.50 F 0.06; P b 0.01), clearly showing the unsettling between beta- and alpha-secretase activity in AD patients. Short-term treatment with ChEI donepezil rescues secretases’ system in AD patients

ADAM10/actin levels was observed (Fig. 2A; mean F SEM ADT0: 0.39 F 0.05, ADT30: 0.73 F 0.13; P b 0.05). In parallel, the release of sAPPalpha significantly increased (Fig. 2B; mean F SEM ADT0: 1.1 F 0.2; ADT30: 1.5 F 0.2; P b 0.05) when compared with baseline, as an index of rescued alphasecretase activity. Accordingly, the immunoreactivity of the 12 kDa molecular weight band CTF99 was visibly decreased in platelets of treated AD patients when compared to T0 conditions of the same patients (Fig. 2C bottom left; mean F SEM ADT0: 155.7 F 19.4, ADT30: 102.5 F 13.8; P b 0.05). Using the same approach as reported above, the ratio between the immunoreactivity of CTF99 and the immunoreactivity of CTF83 decreases significantly in treated AD patients when compared with baseline, reaching levels comparable to CON (Fig. 2C bottom right; mean F SEM ADT0: 0.50 F 0.06, ADT30: 0.26 F 0.03; P b 0.001).

When ADAM10 immunoreactivity was analysed in the same AD patients after 30 days of ChEIs treatment, a rescue of Discussion Degeneration of cholinergic basal forebrain neurons innervating the cortex is believed to contribute substantially to cognitive decline in AD and, thus, it was considered as a promising pharmacological target for ChEIs (Francis et al., 1999). Nevertheless, various questions remain unanswered in the scenario of ChEIs efficacy. Of fundamental interest is whether this class of drugs may have an impact on the natural history of AD, thus proving themselves effective in modifying key pathogenic cascades such as amyloid processing (Liu et al., 2002; Pakaski et al., 2000). Fig. 2. ChEI Donepezil rescues the secretases’ system in platelets of AD patients. (A) ADAM10 and actin in platelets of control subjects (CON) and AD patients at baseline (ADT0) and after 30 days of ChEIs therapy (ADT30). Top: two representative WB analyses of the metabolically active form of ADAM10 (open arrowhead, 68 kDa apparent MW) and the internal standard actin (black arrowhead, 48 kDa) in two control subjects (CON 3, CON 4) and two AD patients both at baseline and at T30 are shown (AD 3, AD 4). Bottom: quantitative analysis: ChEIs treatment causes a significant increase in the ADAM10/actin ratio at 30 days follow up (ADT30) when compared to baseline (ADT0) ( P b 0.05), rescuing it to control levels (ADT30 vs. CON, P = 0.69). (B) Release of sAPPalpha from activated platelets in control subjects (CON) and AD patients at baseline (ADT0) and after 30 days of ChEIs therapy (ADT30). Top: two representative WBs of sAPPalpha release from thrombin activated platelets in two control subjects (CON 3, CON 4) and two AD patients at T0 and T30 (AD 3, AD 4). Bottom: quantitative analysis: the release of sAPPalpha from activated platelets is shown as the ratio of thrombin-provoked release versus basal release T/B. ChEIs treatment enhances significantly sAPPalpha release from platelets of AD patients after treatment for 30 days (ADT30) when compared to baseline (ADT0) ( P b 0.05), rescuing it to control levels (ADT30 vs. CON, P = 0.67). (C) CTF99 and CTF83 in platelets of control subjects (CON) and AD patients at baseline (ADT0) and after 30 days of donepezil therapy (ADT30). Top: two representative WB analyses of CTF99 and CTF83 (12 and 10 kDa apparent MW, respectively) in two control subjects (CON 3, CON 4) and two AD patients before (ADT0) and after 30 days (ADT30) of donepezil treatment (AD 3, AD 4). Bottom left: quantitative analysis: ChEIs treatment causes a significant decrease in the immunoreactivity of CTF99 at 30 days follow up (ADT30) when compared to baseline values (ADT0) ( P b 0.05)—similar to the immunoreactivity of control subjects (ADT30 vs. CON, P = 0.56). Bottom right: quantitative analysis: ChEIs treatment causes a significant decrease in the CTF99/ CTF83 ratio at 30 days follow up (ADT30) when compared to baseline values (ADT0) ( P b 0.001)—similar to the ratio observed in control subjects (ADT30 vs. CON, P = 0.2).

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Our results show in vivo in peripheral cells of AD patients that a short-term treatment with a low dose of the ChEI donepezil is capable of restoring the balance between alpha- and beta-secretase activity altered in AD pathology. In fact, in two previous studies, we have demonstrated that in platelets of AD patients the secretases’ system was modified when compared to controls. Specifically, we observed a reduced in vivo alpha-secretase activity and an altered expression of BACE, the beta-secretase site cleaving enzyme in AD patients (Colciaghi et al., 2002, 2004) in agreement with other studies (Baskin et al., 2000; Li et al., 1995). Here, we confirm and expand these results, demonstrating an unsettling between alpha- and beta-secretase activity in AD patients in vivo, with the beta-secretase activity being predominant as assessed by the ratio between the cleavage products of theses enzymes (CTF83 and CTF99 for the alpha- and beta-secretase metabolite, respectively). A 30-day treatment of donepezil is capable of rebalancing this ratio in vivo. How can donepezil exert this effect? It was already known that ChEIs influence the metabolism of APP in in vitro systems through activation of cholinergic receptors (Nitsch et al., 1992; Pakaski and Kasa, 2003; Racchi et al., 2001, 1999). More recently, we have shown that ChEIs act promoting the trafficking of ADAM10 to the cellular membrane thus increasing its activity in a cholinergic-independent manner (Zimmermann et al., 2004). This aspect of ChEIs mechanism of action easily reconciles the effect we observe in platelets since these circulating cells do not possess a relevant number of cholinergic receptors, although they moderately express acetyl ChE (Zimmermann et al., 2004). Results obtained in this study further reinforce the use of platelets as peripheral cells expressing reliable biomarkers for AD. In fact, we show here that biomarkers of APP metabolism are remarkably influenced by exposure to ChEIs treatment, with their pattern being shifted towards the non-amyloidogenic pathway. This implies the possibility of evaluating dynamically in an intact cell as platelets all steps of APP metabolism. Since we have previously demonstrated that alterations of APP metabolites measured in platelets reflect what is occurring in the central nervous system (Colciaghi et al., 2002), the use of this approach to follow the effect of ChEIs in patients appears a logical consequence. The role of platelets is further consolidated by the observation that CSF markers did not show any variations upon pharmacological treatment (Parnetti et al., 2002) and are, hence, not of use to trace drug response. These findings suggest a positive interaction of ChEIs treatment with pathogenic mechanisms of AD and might explain the clinical effect in the long-term treatment reported with these compounds (Rogers and Friedhoff, 1998; Giacobini, 2001). To present, the efficacy of ChEIs therapy has been clinically proved in randomised, double-blind, placebo controlled, parallel group studies measuring performance based tests of cognitive function, activity of daily living and behaviour (Rogers and Friedhoff, 1996, 1998; for review, see Birks and Melzer, 2000). Also, Positron Emission Tomography studies have been performed to evaluate treatment efficacy (Nordberg, 1999, 2003). A test considering biochemical markers already included in the diagnosis of AD at an early stage to assess the biological efficacy of the drug treatment is therefore of high priority. In fact, the issue of long-term benefit of ChEIs on natural history of the disease is still matter of debate (Courtney et al., 2004). Further, it is unclear if ChEIs exert different effects depending on the stage of the disease and if these drugs limit

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disease progression in early stages when neurodegeneration processes are not yet fully established. Though considering all the possible constraints and evaluating our findings with the necessary caution, our data still demonstrate – in AD patients – that ChEIs are capable of resettling the alterations of the secretases’ system, thus proving themselves effective at a molecular level and demonstrating that a pharmacological treatment can influence the key pathogenic features responsible for AD.

Acknowledgments The authors are in debt to Roberta Epis for skilful technical support in assessing CTF analysis. This study was supported by grants from Ministero Sanita` and COFIN-MIUR 2002 to Monica Di Luca and Progetti Alzheimer Regione Lombarda to Flaminio Cattabeni and Monica Di Luca.

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