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Micro-FTIR reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains Nuria Benseny-Cases, Oxana Klementieva, Marine Cotte, Isidre Ferrer, and Josep Cladera Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Nov 2014 Downloaded from http://pubs.acs.org on November 22, 2014
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Analytical Chemistry
Micro-FTIR reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains Núria Benseny-Cases*,º,∆, Oxana Klementievaʃ, ǂ,ґ, Marine Cotte*,Φ, Isidre Ferrerʃ, ǂ, Josep Cladera§ *European Synchrotron Radiation Facility, 71 avenue des Martyrs, F-38000 Grenoble, France. ʃ Institut de Neuropatologia, IDIBELL-Hospital Universitari de Bellvitge, Universitat de Barcelona, Hospitalet de Llobregat, Spain. ǂ Barcelona; CIBERNED (Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas). Spain. Φ LAMS (Laboratoire d’Archéologie Moléculaire et Structurale) UMR-8220, 3 rue Galilée 94200 Ivry-sur-Seine, France. § Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Centre d’Estudis en Biofísica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Amyloid peptides are the main component of one of the characteristic pathological hallmarks of Alzheimer’s disease (AD): senile plaques. According to the amyloid cascade hypothesis, amyloid peptides may play a central role in the sequence of events that leads to neurodegeneration. There are, however, other factors, such as oxidative stress, that may be crucial for the development of the disease. In the present paper we show that it is possible, by using FTIR Microscopy, to co-localize amyloid deposits and lipid peroxidation in tissue slides from patients affected by Alzheimer’s disease. Plaques and lipids can be analyzed in the same sample, making use of the characteristic infrared bands for peptide aggregation and lipid oxidation. The results show that in samples from patients diagnosed with AD, the plaques and their immediate surroundings are always characterized by the presence of oxidized lipids. As for samples from non-AD individuals, those without amyloid plaques show a lower level of lipid oxidation than AD individuals. However, it is known that plaques can be detected in the brains of some non-AD individuals. Our results show that, in such cases, the lipid in the plaques and their surroundings displays similar oxidation level as the tissues with no plaques. These results point to lipid oxidation as a possible key factor in the path that goes from showing the typical neurophatological hallmarks to suffering from dementia. In this process, the oxidative power of the amyloid peptide, possibly in the form of non-fibrillar aggregates, could play a central role.
INTRODUCTION Alzheimer’s disease (AD) is one of the most common dementias associated with severe cognitive decline and memory impairment. The main pathological hallmarks of the disease in the affected central nervous system are senile plaques (SP), neurofibrillary tangles (NT) and cell death.1, 2 Since the socalled amyloid peptide was postulated as the main component of SPs and tau as the main component of NTs, the scientific community has strived to find a link between amyloid deposits, NT and cell toxicity.3-6 What triggers the disease and what determines its development remains unresolved. However, the dominant view nowadays is based on the so-called amyloid cascade hypothesis, by which the amyloid peptide would play a central role in AD. Nevertheless other important physico-
chemical factors have been identified in affected brains that seem to be tightly related to the pathology. For example, the onset and development of AD has been associated with oxidative stress in many systems: cultured cells overexpressing Aβ, C.elegans models, transgenic mice and post-mortem human samples showing increased levels of oxidized lipid, protein and DNA.7-12 Moreover, Aβ has been described as a lipid oxidant agent in vitro13 and, on top of that, amyloid plaques are rich in oxidizing metals such as Cu, Zn and Fe.14 Additionally, there is some controversy as to whether the Aβ-Cu complexes are toxic or, conversely, whether the Aβ-Cu complex formation may be a protection mechanism against the oxidative capacity of the metal. Many authors suggest that the neurotoxicity of Aβ peptides is due to the induction of oxida-
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Analytical Chemistry
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tive stress associated with the redox cycle of copper ions bound to Aβ peptides.15-17 A vast number of studies using homogenates of ADaffected human tissue have shown an alteration of the mitochondrial electronic transport chain, as well as oxidation of structural proteins, chaperones and proteins of the ubiquitinproteosome system. In relation to lipid oxidation, 4Hydroxynonenal (4-HNE), a derivative of lipid and carbohydrate oxidation, has been commonly used as a marker, and inmunohistochemical analysis of brain tissue has shown weak localization of 4-HNE in plaques.18 It is well known that amyloid deposits are associated with lipid membranes, that lipid membranes can alter Aβ structure and that Aβ can damage membranes.19 Moreover, lipid membranes are among the components most vulnerable to oxidative stress.20, 21 However, detecting and studying lipid peroxidation directly in the tissue (post-mortem samples) is a difficult task because of the interference caused by the variety of chemical components present. Spectroscopic methods, such as the measurement of diens absorption at 234 nm for example, largely used in vitro, cannot be applied in tissue due to the high background UV absorption. Chemiluminescent methods are also used in vitro but they are difficult to apply in tissue due to the low intensity of the biological matrices.22 In recent decades, Fourier-Transform Infrared (FTIR) spectroscopy has emerged as a good technique for the measurement of lipid oxidation.23, 24 The absorption of the carbonyl group of the ester bond, which can be monitored in the infrared spectrum at 1740 cm-1, may increase as a consequence of lipid oxidation. In addition, free radicals, the product of lipid oxidation, can react with each other, leading to the generation of a double bond that will increase the absorption of the C=C-H group at 3012 cm-1 with a corresponding decrease in the absorption of C-H bonds at 2920 cm-1 .25-27 FTIR also permits the study of the secondary structure of proteins together with the characterization of lipid oxidation. The technique is especially indicated for the study of amyloid deposits, where the accumulation of protein implies a change in the protein secondary structure, from unordered and α-helix into characteristic β sheet structures. The study of proteins and lipids in tissue slides using FTIR spectroscopy is possible due to the existence of microscopy techniques such as microFTIR (µFTIR), which allows the spatial study of specific bands with high resolution and sensitivity, and, when used with a synchrotron radiation (SR) source, may permit a spatial resolution of 6 µm.28-32 In the present work we have aimed to characterize the oxidation state of the lipidic component in relation to the presence of senile plaques in tissue samples from AD brains. EXPERIMENTAL SECTION Human samples. Human brain tissue was obtained from the Institute of Neuropathology Brain Bank (HUB-ICOIDIBELL Biobank) following the guidelines on this matter of the Spanish legislation and of the local ethics committees. One hemisphere was immediately cut in coronal sections, 1 cm thick, and selected areas of the encephalon were rapidly dissected, frozen on metal plates over dry ice, placed in individual air-tight plastic bags, numbered with water-resistant ink, and stored at -80°C. The other hemisphere was fixed by immersion in 4% buffered formalin for 3 weeks for morphological study. The neuropathological categorization was performed according to the Braak and Braak nomenclature adapted for paraffin sections.33 Cases with associated neuro-
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degenerative processes (i.e. Lewy pathology, argyrophilic grain pathology) and metabolic syndrome were not included in the present series. Cases with systemic inflammatory (including autoimmune) and infectious diseases were rejected. Special care was also taken not to include cases with prolonged agonic state. The post-mortem interval between death and tissue processing was between 3h and 18h. The following cases were used in the present study: AD-related pathology Braak and Braak stages I/0 (two men aged 64 and 71 years), AD-related pathology stage II/A (one man aged 88 years), AD-related pathology stage III/B (two women aged 79 and 75 years), AD stage V/C (one man aged 93 and one woman aged 79). The parietal cortex was used for the present studies. Cases with stage 0 had no β-amyloid plaques, stage A had a few diffuse plaques in the parietal cortex, stage B had both diffuse and neuritic plaques, and stage C large numbers of diffuse and neuritic plaques in the parietal cortex. Immunohistochemical studies disclosed that diffuse plaques were composed of amyloid 1-42 whereas neuritic plaques were composed of βamyloid 1-42 and β-amyloid 1-40. The control found with plaques corresponds to one man aged 64 diagnosed with amyloid deposits and neurofibrillary tangles. Preparation of human brain tissue for synchrotron FTIR mapping. Frozen brain sections of the parietal cortex, 6 µm thick, were cut on a Leica CM3050S cryostat (Leica Microsystems, Wetzler, Germany) at -18° C thaw-mounted onto CaF2 or BaF2 slides (Crystran, UK), fixed in freshly prepared PBS buffered 4% paraformaldehyde for 10 min at room temperature and stored at -20° C until use.34
Figure 1. Typical FTIR spectra (grey) and second derivative spectra (black) of a tissue where the important bands are labelled: C-H area corresponds to the hydrophobic chain of the phospholipid where the unsaturated C-H band can be clearly distinguished from the saturated C-H band. As well, the CH3 terminus has been labelled in the C-H area. In the amide I region, the β sheet structure absorbs at 1630 cm-1 while the α-helix structure absorbs at 1650 cm-1. COOH absorption is also labelled in the graph at 1740 cm-1.
Immunohistochemistry staining. 15 µm thick brain tissue of the parietal cortex sections obtained with a cryostat were thaw-mounted on Thermo Scientific™ Polysine adhesion slides (Thermo Scientific, Germany) and fixed in 4% paraformaldehyde for 10 min. Slides were stored at -20° C until use. Before the immunohistochemical staining, the slices were warmed in a desiccator at room temperature, immersed in
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Analytical Chemistry
Figure 2. Aβ Immunostaining of (a) ADII, (e) ADIII and (i) ADV brain samples; (b, f, j) 6E10 staining of Aβ and (c, g, k) ThS (specific for fibrils) staining, of the a ADII, ADIII and ADV brain samples, respectively; (d, h and l) 6E10 and ThS staining superimposed; (m) ThS/Aβ ratio as a measure of the fibrillarity level of the plaque (*p
