Biological markers in Alzheimer disease

Neurobiology of Aging 24 (2003) 191–193

Meeting report

Biological markers in Alzheimer disease夽 Carlo Ferrarese∗ , Monica Di Luca Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, Via Donizetti 106, 20052 Monza (MI), Italy Received 25 March 2002; accepted 27 March 2002

Keywords: Alzheimer disease; Biological markers; Amyloid precursor protein; Mitochondrial DNA; Oxidative stress; Glutamate; Excitotoxicity; Caveolin; Inflammation; Cytokines; Chemokines; Platelets; Fibroblasts

Diagnosis of Alzheimer disease (AD) is based on clinical features and confirmed only at pathological brain examination. However, since markers of pathogenic mechanisms have been demonstrated in biological fluids and in peripheral tissues, several of them have been proposed as in vivo diagnostic tools. Their importance is underscored by the need to discriminate among various types of dementia and by the evidence that effective treatments should start at early phases of disease or even at preclinical stages. Apart from genetic mutations responsible for the few cases of familial AD, so far no single biological test has reached a reliable degree of sensitivity and specificity for a definite in vivo diagnosis, to detect susceptible individuals at preclinical stage or patients suitable for appropriate drug therapies. More recently, however, combinations of various biological markers have shown higher reliability as diagnostic tools. For these reasons the research on biological markers in AD is presently blooming and the Italian Interdisciplinary Network on Alzheimer Disease (ITINAD, www.itinad.com), a scientific team of clinicians and researchers, which brings together neurologists, psychiatrists, geriatricians, pharmacologists, and molecular biologists, devoted its second monothematic symposium to this theme. The first part of the meeting covered the results of a multi-center study, sponsored by the Italian Ministry of Education and Research, which investigated the interactions of various pathogenic events (amyloid processing, mitochondrial dysfunction, oxidative stress, excitotoxicity) in peripheral tissues obtained from AD patients. 夽 Report of the International Symposium, sponsored by the Italian Interdisciplinary Network on Alzheimer Disease (ITINAD), University of Milano-Bicocca-Monza (Italy), November 3, 2001. ∗ Corresponding author. Tel.: +39-233-3598; fax: +39-233-3586. E-mail address: [email protected] (C. Ferrarese).

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In the introductory lecture, Ashley Bush, Associate Professor of Psychiatry at Harvard Medical School, Boston, described mechanisms of amyloid toxicity, pointing out the role of metal ions and free radicals in the pathogenesis of Alzheimer’s disease. Metal ions like copper and zinc are enriched in amyloid deposits of AD patients’ brains. Bush presented various studies about the amyloid reducing effect of clioquinol, a bioavailable copper/zinc chelator, in APP2576 transgenic mice. His positive results support the use of drugs with metal-binding properties as a novel approach for the prevention and treatment of Alzheimer’s disease [1]. The following three talks discussed platelets and fibroblasts as easily accessible peripheral biomarkers of altered amyloid processing in AD. Alessandro Padovani, Professor of Neurology at the University of Brescia, spoke about platelet amyloid precursor protein (APP) isoforms in AD patients. An altered pattern of APP forms, consisting in a reduced ratio between the optical density of the upper (130 kDa) and the lower (106–110 kDa) immunoreactivity bands, has been described in platelets of patients with AD. Padovani’s study evaluated the sensitivity and the specificity of the ratio of platelet APP forms as a marker for AD: analyzing the ratios in patients affected by this disease and comparing them to the ratio pattern of control subjects or patients with mild cognitive impairment (MCI), revealed that the platelet APP ratio allows to differentiate AD from both normal aging and other dementing disorders with high sensitivity and specificity [11,12]. Monica Di Luca, Professor of Neuropsychopharmacology at the University of Milan, characterized the platelet APP processing and presented the various APP metabolites and secretases in AD platelets. APP can be processed by alpha-secretase (whose most putative candidate is ADAM 10) in a nonamyloidogenic pathway, leading to the release of soluble alpha-APPs. The other—mutually exclusive— pathway, which includes beta- and gamma-secretase,

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liberates the amyloidogenic component of APP. ADAM 10 is a N-glycoprotein and its protein levels are remarkably decreased in AD platelets compared to controls (Colciaghi et al., in press). Antibodies recognizing both the Nand the C-terminal domain of BACE, a candidate holding beta-secretase activity, reveal two specific bands at about 53 and 34 kDa apparent molecular weight, suggesting the existence of two different splicing forms of the protein in platelets, a hypothesis which was confirmed by RT-PCR experiments. The 34 kDa band was obtained in western blot experiments of control subjects and in mild AD patients as well, whereas severely affected patients showed markedly decreased level of the protein; this suggests a possible role of the lower BACE splicing form in the molecular pathogenesis of AD (Colciaghi et al., in preparation). The first part of the morning session was concluded with a talk about the processing of fibroblast APP in AD and Down syndrome (DS). Marco Racchi, Professor of Pharmacology at the University of Pavia, discussed skin fibroblasts derived from patients as a useful peripheral tissue to study altered processing of APP in AD and DS. The reported data showed the possibility to exploit the biochemical differences identified in AD patients versus control subjects with the aim to produce a specific biological indicator, able to distinguish AD and control fibroblasts. It is possible to characterize a discriminating scale by considering a series of biological alterations correlated to APP metabolism and its regulation, which appear in cellular lines of AD and DS patients. The scoring system presented allows the generation of an index distinguishing AD from controls on a biological point of view (unpublished data). Altered amyloid processing may interfere with mitochondrial functions and various authors reported impaired activity of mitochondrial enzymes in AD, not only in brain, but in peripheral tissues as well. Nereo Bresolin, Professor of Neurology at the University of Milan, demonstrated the progressive accumulation of mutations of the D-loop region of mitochondrial DNA with aging [2]. In the framework of this project he found a specific T414G point mutation in mitochondrial DNA extracted from fibroblasts of AD patients and early appearance of mutations in this region in DS patients. Impaired mitochondrial activity and amyloid itself may trigger oxidative stress in various cell types. Marzia Galli-Kienle, Professor of Chemistry at the University of Milano-Bicocca, investigated plasma levels of thiobarbituric reactive substances (TBARS), markers of lipid peroxidation, before and after oxidative stress induced by copper ions, in AD patients, MCI patients, and age related normal controls. Basal TBARS levels were slightly but not significantly higher in plasma from AD and MCI patients, while increased up to three times, respect to control patients, after oxidative stress [4]. Thus, increased oxidative stress susceptibility of plasma lipids may represent a peripheral marker of pro-oxidant activity of beta-amyloid. Adducts of A␤ peptides with Hydroxynonenal (HNE), one of the end

products of the lipidoperoxidation process, were also identified and characterized by LC-ESI-MS [9]. Plasma membrane lipids have been extensively studied by Marina Pitto, Associate Professor of Biochemistry at the University of Milano-Bicocca, who demonstrated colocalization of APP and caveolin in lipid rafts prepared from AD fibroblasts [13]. Alterations of lipid metabolism in AD peripheral cells has been also demonstrated, which may be linked to systemic alterations of APP processing. Carlo Ferrarese, Professor of Neurology at the University of Milano-Bicocca, has demonstrated the possibility to use peripheral models, such as platelets and fibroblasts, to investigate alterations of glutamate transport and the role of excitotoxicity not only in AD [6], but also in other neurodegenerative disorders, such as Parkinson disease [5] and amyotrophic lateral sclerosis [7]. Slow excitotoxicity, triggered by chronic oxidative stress and damage of glutamate transporters, appears as a final common mechanism leading to neuronal apoptosis in neurodegenerative disorders. Fibroblast cell lines prepared from AD patients may be useful models to investigate the specific mechanisms that link altered APP processing to glutamate alterations in this disease. In fact, glutamate uptake is impaired in fibroblasts from AD and DS patients, is more sensitive to the effect of products of lipid peroxidation, such as HNE, and is restored by treatment with anti-oxidants such as glutathione. Specific glutamate transporter alterations seem to mediate these effects. Moreover, preliminary results presented by Professor Ferrarese at the symposium indicate that these events may appear early in course of the disease and that MCI patients already present decreased platelet glutamate uptake. Kaj Blennow, Professor of Neurology at the University of Goteborg (Sweden), closed the morning session with a lecture covering the studies performed to confirm the specificity and sensitivity of biomarkers such as sAPP, beta-amyloid, and Tau in cerebrospinal fluid of AD patients and different neurological disorders. Combined amyloid and Tau measurements may provide higher sensitivity and specificity, as demonstrated in large scale studies, performed in routine clinical assessments in neurological departments all over Sweden. The second part of the symposium was focused on peripheral markers of inflammation in AD. Joseph Rogers, President and Senior Scientist of the Sun Health Research Institute in Sun City, AZ, USA, opened the session with a lecture on inflammatory mechanisms in AD. Rogers’ group developed microglial and astrocyte cultures derived from brains of AD and elderly control patients obtained few hours after death. With continued exposure to A␤, cultured microglia become progressively more activated, and show increased expression of various inflammatory mediators, such as IL-1, IL-6, and TNF-␣; chemokines complement component C1q, the growth factor M-CSF and reactive nitrogen intermediates. Rogers’ laboratory has recently focused on transcription factors, belonging to C/EBP family, that are involved in regulating inflammatory

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mediators. C/EBP-␤ expression was found significantly increased in AD cortex, particularly by astrocytes surrounding A␤ deposits. Similarly, C/EBP-␤ is upregulated in the AD cortex and is highly expressed by microglia within A␤ deposits. Nonsteroidal anti-inflammatory drugs (NSAIDS) may be an ideal therapeutic adjunct to A␤ removal approaches, because they don’t hinder phagocytosis of A␤, while inhibiting secondary inflammatory toxicity to nearby cells. M.E. Luigi Grimaldi, Head of Department of Neuroscience, Caltanissetta Hospital, pointed out the link between cytokine gene polymorphisms and AD age at onset. The TT genotype of IL-1␣ is associated with an almost 10 years earlier onset of AD compared to the C/C genotype. Recently, IL-1␤ + 3953 polymorphism seems to be associated with anticipation of age at onset of AD. Furthermore, different IL-6 polymorphisms influence IL-6 plasma levels in AD [10]. To search for a bio-marker useful to differentiate AD from other dementing diseases, Elio Scarpini, from the Department of Neurological Sciences of Ospedale Maggiore Policlinico, Milan, proposed to evaluate chemokine levels in CSF and serum. Preliminary data showed a modification of IP-10 and MCP-1 levels in demented patients, compared to age-matched healthy controls. Maria Grazia De Simoni, from Department of Neuroscience of the Mario Negri Institute for Pharmacological Research in Milan, investigated plasma cytokine levels and cytokine release after lipopolysaccharide (LPS) stimulation in demented patients. A significant increase in circulating TNF-␣ in multi-infarctual dementia (MID) patients was found, while TNF-␣, IL-6, and IL-10 release were significantly reduced in AD and MID patients respect to controls [3]. Similar results were shown by Gloria Galimberti, from Department of Neuroscience and Biomedical Technologies of the University of Milano-Bicocca, who demonstrated a decrease in cytokine release, after LPS stimulation, in severe AD patients versus controls. On the contrary, IL-1␤ and TNF-␣ release were found significantly increased in MCI patients. This data suggest increase in immune response in preclinical subjects, followed by a progressive down regulation, maybe triggered by chronic A␤ stimulation [8]. A round table, chaired by Vincenzo Bonavita and Nicolò Rizzuto, respectively former and present Presidents of the Italian Neurological Society, closed the symposium with an overview of specific patterns of biochemical modifications demonstrated in peripheral fluids and tissues of AD patients. Such peripheral markers may be proposed for in vivo investigation of pathogenetic mechanisms, for early or preclinical diagnosis and to address specific therapies. In

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