Transferrin neutralization of amyloid β 25–35 cytotoxicity

Clinica Chimica Acta 350 (2004) 129 – 136 www.elsevier.com/locate/clinchim

Transferrin neutralization of amyloid h 25–35 cytotoxicity Sergio Giuntaa,*, Roberta Galeazzia, M. Beatrice Vallia, Elizabeth H. Corderb, Luciano Galeazzia a

Laboratorio Analisi Chimico-Cliniche, Microbiologiche e Diagnostica Molecolare, Ospedale Geriatrico INRCA (IRCCS), via della Montagnola 81, 60100 Ancona, Italy b Center for Demographic Studies 2117 Campus Drive, Box 90408, Duke University, Durhan, NC 27708-0408, USA Received 28 April 2004; received in revised form 10 July 2004; accepted 13 July 2004

Abstract Background: Fibrillar aggregates of amyloid h 25–35 (Ah25–35) form rapidly in vitro able to lyse human red blood cells (RBCs). Human sera, albumin, and apolipoprotein E (ApoE) each limit fibrillation and cytotoxicity. Potentially, these substances protect neurons from Ah1–40/42 aggregates. Transferrin (TF) is investigated in this study. Methods: The Mattson red blood cells model was employed to determine whether co-incubation of transferrin and Ah25–35 prevented lysis. The formation of fibrillar Ah25–35 in the presence of transferrin was investigated using Congo red staining and spectrophotometric studies. Results: We found that incubation of 20 AM Ah25–35 with physiologic levels of transferrin prevented red blood cells lysis and the formation of macro-aggregates. Conclusions: These in vitro results suggest that transferrin may limit fibrillar h amyloid formation in vivo and cytotoxicity. D 2004 Published by Elsevier B.V. Keywords: Transferrin; Amyloid h; Alzheimer pathogenesis; Red blood cells

1. Introduction

Abbreviations: ApoE, apolipoprotein E; AhPP, amyloid hprecursor protein; AD, Alzheimer’s disease; Ah25–35, amyloid h fragment 25–35; Ah1–42/43, amyloid h fragment 1–42/43; TF, transferrin; RBCs, red blood cells; CSF, cerebrospinal fluid; O.D., optical density. * Corresponding author. Tel.: +39 71 8003394; fax: +39 71 8003343. E-mail address: [email protected] (S. Giunta). 0009-8981/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.cccn.2004.07.025

Ah peptides are fragments of amyloid h-precursor protein (AhPP) following cleavage of membranebound AhPP to soluble AhPP fragments, varying in length and propensity to form fibrils [1]. Deposits of Ah1–40/42 fibrillar aggregates in the brain as senile plaque and as amyloid angiopathy are among the key pathological hallmarks of Alzheimer’s disease (AD) [1]. Free iron is known to catalyze the production of

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oxygen-free radicals and formation of Ah fibrillar aggregates [2–4]. Ah1–40/43 is amphiphilic with a hydrophilic Nterminus (residues 1–28) and a hydrophobic Cterminus (residues 29–39 or 29–43). The 11-residue amyloid h 25–35 (Ah25–35) fragment of amyloidogenic Ah1–40/42 retains activities of the full-length peptide rapidly forming fibrillar aggregates highly cytotoxic to neuronal cells. The four N-terminal polar residues of the 11-residue Ah25–35 fragment include a charged lysine residue (the net charge is +1), the seven C-terminus residues are predominantly hydrophobic, and the single-letter amino acid sequence is +H3N-GSNKGAIIGLM-COO . The hydrophobic segment corresponds to a part of the transmembrane domain of AhPP [5]. This hydrophobic [6] 11-residue of Ah25–35 fragment of Ah1–40/ 42 rapidly forms fibrils that lyse human red blood cells (RBCs) [7–9]. The fibrillar aggregates resemble Ah1–40/42 aggregates, and both in vivo and in vitro are highly cytotoxic to neuronal cells as well as lytic for human RBCs in vitro [7]. Iron has been implicated in the fibrillar aggregation of Ah25–35 [3]. These properties suggest that preincubation of Ah25–35 with test substances may be able to prevent RBC lysis and identify inhibitors of toxicity [7,8,10]. Prevention of RBCs lysis may facilitate preliminary identification and screening of these natural and synthetic substances for their ability to protect against fibrillar Ah25–35 damage. Human serum completely prevents RBCs lysis, and demonstrated protective components are apolipoprotein E (ApoE) and albumin [8,10]. Transferrin (TF) is a plasma 78,000 m.w. glycoprotein with two Fe(III) binding sites each with a high affinity for Fe3+ of 1021 M 1 needed for the transport of iron [11]. It is synthesized primarily in the liver, but significant amounts are also produced in the brain [12]. TF delivers iron to the brain across the blood– brain barrier via specific receptors located on the brain microvasculature [13]. Bloch et al. [14] showed that brain contains TF and TF messenger RNA (TF mRNA). Brain levels of TF mRNA (highest in oligodendrocytes and the choroid plexus) approximate liver levels [15]. However, there may be a functional difference between brain TF and in other tissues such as liver and a specific role of TF in oligodendrocyte maturation and in myelinogenesis [16].

Judging from the concentrations of iron and TF in cerebrospinal fluid (CSF) from control humans, it is estimated that CSF TF is fully saturated with iron, and that nontransferrin-bound iron is also present [17]. The presence of nontransferrin-bound iron in brain extracellular fluid, including CSF, has been also reported by Moos and Morgan [18] and by Lipscomb et al. [19]. In end-stage AD, a dramatic reduction of TF in the CSF has been demonstrated [20]. It is well known that h amyloid peptide 1–42 concentrations in the CSF are significantly lower in AD patients compared to age-matched controls [21]. These findings suggested a possible role for TF in AD pathogenesis and as a test compound in the RBC lysis test of amyloid h toxicity [7]. We have utilized this model for screening natural and synthetic compounds useful in the identification of substances able to protect against amyloid h cytotoxic effects. We examined the possibility that TF, present in human serum, CSF, and brain can prevent Ah25–35 fibril aggregates formation and RBCs lysis in vitro. We found that physiologic quantities of TF were highly protective against RBCs lysis and prevent the formation of fibrillar aggregates of Ah25–35.

2. Materials and methods Synthetic Ah25–35 was purchased from Sigma (St Louis, MO, USA). A stock solution (1 mM in distilled water) was prepared 2 h prior to experiments. Ah25–35 is known to form fibrils immediately after solubilization [22]. Thus, after 2 h, the stock bsolutionQ would consists of Ah25–35 fibrils. Human TF was purchased from Sigma. A stock solution (1 mg ml 1 in distilled water) was prepared each day prior to experimentation. Congo red was purchased from Sigma. A stock solution (8.6 mM in distilled water) was prepared immediately prior to experiments and protected against light exposure. 2.1. Preparation of RBC RBCs were obtained from fresh heparinized blood samples from young adult volunteers by centrifugation at 500g for 10 min. The pellet was washed three times with four volumes of

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Locke’s solution (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, and 5 mM HEPES; pH 7.2). The buffy coat and a portion of the upper layer of RBCs were removed by aspiration at each wash. The remaining RBCs were resuspended in four volumes of Locke’s solution. 2.2. Lysis experiments Experiments were preformed in triplicate in 5-ml plastic test tubes (Bio Merieux Italia, Rome, Italy). Each tube contained ~250,000 erythrocytes suspended in 1 ml Locke’s solution. Experiment 1: RBCs were exposed to varying concentrations of Ah25–35 (0, 5, 10, 20 AM) with and without CSF levels of TF [15-min preincubation performed after the 2 h required for Ah fibrillation, final concentration of TF was 6 Ag/ml (7.6 10 5 mM) (equivalent to normal CSF concentration of TF)]. Experiment 2: 10 and 20 AM Ah25–35 were preincubated for 15 min with microvolumes containing a range of TF concentrations in order to investigate the quenching of lysis. The upper range of Ah25–35 concentration was set below 50 AM known to lyse all erythrocytes. Tubes were incubated at room temperature for 4–6 h to allow time for lysis. Thereafter, tubes were centrifuged to pellet remaining RBCs. Optical absorbance meas-

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urements of the supernatant were made at 540 nm (Uvikon 860 spectrophotometer, Kontron-Milan, Italy). The optical density (O.D.) of the supernatant was compared (within the linear range) to that found for an equivalent number of cells lysed by exposure to 0.1% sodium dodecyl sulfate (SDS) and multiplied by 100 to arrive at the percent lysis. Neither SDS nor TF interfered with absorbance measurements at the wavelength used or made the samples turbid. 2.3. Congo red experiments We adapted [10] the method of Klunk et al. [23] and Wood et al. [24] devised to stain Ah1–42 fibrils. A final concentration of 25 AM Congo red was added to 1 ml of 50 mM phosphate buffer (pH 7.4). There were two negative controls consisting of Congo red with or without 12.5 Ag/ml TF, a positive control consisting of Congo red+20 AM Ah25–35, and the test combination of Congo red, 20 AM Ah25–35 pretreated 15 min with 12.5 Ag/ml TF. Spectrophotometric measurements on supernatants after centrifugation were done immediately and at 24 h. The presence of fibrillar amyloid can be detected by a spectrophotometric method; in fact, fibrillar amyloid peptides are known to alter the Congo red spectrum with the point of maximal spectral difference at 540 nm according to a method described by

Fig. 1. Ah25–35 concentration and % RBC lysis (upper line). Co-incubation of Ah25–35 and TF at CSF concentration [6 Ag/ml, 7.610 (lower line), shows that, TF prevented RBC lysis. Experiments were performed in triplicates.

5

mM]

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Fig. 2. TF quenching of Ah25–35-induced lysis at 20 AM (upper line) and 10 AM (lower line). Experiments were performed in triplicates.

Klunk et al. [23] and Wood et al. [24]. Moreover, when fibrillation of h amyloid progressively goes further, micro-aggregates and possibly macro-aggre-

gates are formed; these aggregates are easily visible by the naked eye in tubes as well as in macrotiter wells, and can be also detected by spectrophotometric

Fig. 3. Photographs of macrotiter plates taken at 24 h to demonstrate presence of fibrillar aggregates. (A) Test: 20 AM Ah25–35 preincubated with 12.5 Ag/ml TF. (B) Negative control: 25 AM Congo red. (C) Positive control: Ah25–35 20 AM. (D) Negative control: Congo red+TF.

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studies in the supernatants after centrifugation of the aggregates and detection of residual Congo red optical density.

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10 and 20 AM, pretreatment with subphysiologic levels of TFN210 5 mM prevented RBC lysis (Fig. 2). 3.2. Congo red experiments

3. Results 3.1. RBC lysis experiments We first verified that TF alone did not cause RBC lysis after four to 6 h incubation at room temperature and complete lysis at 50 AM Ah25–35 (data not shown). Indeed, 50 AM Ah25–35 caused 100% lysis equal to the effect of SDS. Experiment 1: In the absence of TF, the percentage lysis increased with Ah25–35 peptide concentration (Fig. 1). There was 27% lysis at 5 AM, 45% at 10 AM, and 76% lysis at 20 AM. Pretreatment with CSF levels of TF [6 Ag/ml (7.610 5 mM)] resulted in near-complete protection against Ah25–35-induced RBCs lysis at each peptide concentration. There was b15% lysis at 20 AM. Experiment 2: At Ah25–35 concentrations of

No visible macro-aggregates were found after coincubation of 12.5 Ag/ml TF and 20 AM Ah25–35 for 24 h, although micro-aggregates were clearly present (Fig. 3A). Without TF, Ah25–35 macro-aggregates were visible to the naked eye within 4–6 h and obvious at 24 h (C). Neither Congo red (B) nor TF alone (D) demonstrated red fibrils. Fig. 4A shows the absorbance spectra at 540 nm at the start of the experiment for 25 AM Congo red alone as a control (1) and Congo red+20 AM Ah25– 35 (2). Fig. 4B superimposes the absorbance spectra for Congo red+20 AM Ah25–35+12.5 Ag/ml TF (3) on curves 1 and 2. Tubes containing the Ah25–35 peptide, with and without TF, demonstrated an increased absorption and a E-shifted Congo red spectra indicating the presence of fibrillar Ah25–35. The control curve of Congo red plus 12.5 Ag/ml TF

Fig. 4. (A) Absorbance spectra at time 0 for 25 AM Congo red (1) and for Congo red+20 AM Ah25–35 (2). (B) Spectra 1 and 2 superimposed with 3 for the test solution also containing 12.5 Ag/ml TF.

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exactly overlapped that of Congo red alone (not shown). After 24 h, each tube was centrifuged to remove macro-aggregates. Absorbance spectra at 540 nm were repeated for the supernatants (Fig. 5). Curve 1 was unchanged for the Congo red control tube. The Congo red+Ah25–35 tube had little residual absorbance (2), that is, the Ah25–35 was found as macro-aggregates. The btestQ tube containing TF retained a higher residual absorbance indicating the presence of fibrillar Ah25–35 microaggregates (3). Thus, 12.5 Ag/ml TF, sufficient to prevent Ah25–35-induced RBCs lysis and formation of large fibrillar aggregates, nonetheless demonstrated micro-aggregates. The reduced optical density for TF+Ah25–35 compared to TF alone (not shown) and sediment after centrifugation also indicate the formation of aggregates large enough to be sedimented.

Fig. 5. Absorbance spectra at 24 h of supernatants after centrifugation to remove large aggregates. The Congo red control curve is unchanged (1). There is little residual absorbance, that is, few micro-aggregates, in the Congo red+Ah25–35 tube (2). (3) The tube also containing TF demonstrated residual absorbance, that is, microaggregates.

4. Discussion Alzheimer’s disease neuropathogenesis is associated with extracellular deposition of amyloid h peptide (Ah) and development of intracellular neurofibrillary tangles composed of microtubule-associated protein tau. Although also tau protein is present in CSF, the majority of published literature on Ahcytotoxicity does not include the tau protein in the experimental set-up. This happens primarily because of the extracellular location of Ah fibrils formation as opposed to the intracellular nature of tau-associated tangles. Indeed, also investigators in these fields are distinguished as bAPTISTs (bAPeptide) and TAUISTS (TAU protein). How these AD features interrelate and determine disease is unclear [25]. In this paper, we investigated Ah-mediated cytotoxicity in vitro in relation to the major iron-binding protein, TF. Free iron catalyzes the production of oxygen-free radicals facilitating, among other unwanted processes, formation of toxic Ah fibrillar aggregates in vitro [2– 4]. Even the trace amounts found in buffers and reagents may determine the bspontaneousQ fibrillation of Ah in vitro [3,4]. To overcome the solubility and toxicity of iron, there are two proteins mostly involved in iron homeostasis. Iron is transported by TF and sequestered by ferritin. Binding to these proteins allows transport and storage in a relatively nonreactive state and thus serves as important antioxidant defense mechanism [26]. Importantly, both proteins are normal brain constituents. An imbalance of brain iron homeostasis may be involved in AD. Patients have lower serum TF levels compared to age-matched cognitively intact control subjects [27], lower than other elderly [28] and/or malnourished [29,30] persons. As AD frequency increases with age and dementia itself is associated with malnutrition, further studies to clarify these issues are needed. Therefore, CSF TF relation to TF serum concentration in the elderly and in AD patients is a complex matter. Also, the blood–brain barrier could play a role; indeed, the pathology of AD is not limited to amyloid plaques and neurofibrillary tangles. Evidence suggests that more than 30% of AD cases exhibit cerebrovascular pathology, which involves the cellular elements that represent the blood–brain barrier [31]. The compelling vascular pathology associated with AD suggests that transient and focal

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breach of blood–brain barrier occurs in late onset AD and may involve an interaction of several factors, which include perivascular mediators as well as peripheral circulation-derived factors that perturb the endothelium. These vascular abnormalities are likely to worsen cognitive disability in AD [31]. Pathologically, brains from AD patients demonstrate accumulation of iron in senile plaques together with an altered distribution of iron transport and storage proteins [32]. The low levels of TF may prevent efficient transport and sequestration of iron [15]. Brain TF levels decrease with age dramatically when AD or Parkinson’s disease are superimposed on the aging process [20]. The ratio of TF to iron, a possible index of iron mobilization capacity, is low in the globus pallidus and caudate putamen in both disorders [15]. Low TF levels is a suggested cause of increased brain iron concentrations in these disorders [33]. There are reports of low brain TF levels in AD [20,33] circumstantially supporting the oxygen radical hypothesis of AD [1–5,27]. Two additional links to AD are the dramatically reduced concentration of TF in the CSF in end-stage AD [20], and the demonstration that TF is homogenously distributed around the senile plaques and is apparently extracellular [33]. These data suggest a role for TF in AD pathogenesis. We must consider that it has already been shown by several authors that the experimental administration of aggregated h amyloid peptide (25–35) shows also in vivo toxicity. The experimental in vivo administration of aggregated h amyloid peptide (25– 35) is associated with oxidative stress in the hippocampus, and with disturbances in ion homeostasis [34]. The in vivo significance or red blood cells lysis by aggregated h amyloid peptide (25–35) has not yet been investigated; however, it is worth of consideration that recently, Jayakumar et al. [35], studying red cell oxidative cell damage by amyloid h protein and perturbation on cell volume, stated bthat their results suggest that amyloid interactions with the red cell may contribute to the pathology of ADQ. In summary, we investigated whether the iron transport protein transferrin was able to prevent fibrillar Ah25–35 formation and red blood cell lysis in vitro. Physiologic levels prevented cytolysis and the formation of macro-aggregates stainable by Congo red (micro-aggregates were found). Our finding

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implies that experimental systems investigating Ahfibrillar aggregate formation and Ah-direct cytotoxicity consider the interaction of TF with Ah. In addition, low levels of TF found in aging may contribute to formation of senile plaques and neuronal cytotoxicity.

Acknowledgements Financial support for the study provided by grants from Italian Ministry of Health and National Institute on Aging (USA). We acknowledge the expertise of Marzio Marcellini and Lucia Montemurro in the reediting of figures.

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