Review
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Novel fluorescent microarray platforms: a case study in neurodegenerative disorders Expert Rev. Mol. Diagn. 13(8), 863–873 (2013)
Marina Cretich*, Laura Sola, Paola Gagni and Marcella Chiari Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy *Author for correspondence: Fax: +39 022 850 1239 [email protected]
This review focuses on the fluorescent protein microarrays applied to neurodegenerative disorders, a major health problem in our aging society. Biomarker discovery studies and work on new diagnostic tests are both included. Three platforms are described: antibody planar microarrays, comprising an array of well-defined antibodies for the simultaneous capture of biomarkers; antigen planar microarrays in which proteins, peptides or small molecules are immobilized and probed with biological samples to chase specific antibodies; and bead-based array platforms, which are flow cytometry-based methods in which capture molecules are coupled to fluorescent microspheres, facilitating the simultaneous quantification of several analytes. KEYWORDS: Alzheimer’s disease • antibody • biomarkers • fluorescence • microarrays • neurodegeneration
Neurodegenerative disorders (NDs) are characterized by a progressive loss of function of specific groups of neurons in different regions of the brain. The major disease groups are Alzheimer’s disease (AD), vascular dementia, Parkinson’s disease (PD), frontotemporal lobar degeneration, Lewy body dementia and amyotrophic lateral sclerosis. These disorders are variably associated with dementia, personality changes, language abnormalities or progressive muscle weakness. Because the greatest risk factor for NDs is age, these diseases are considered major health and quality of life problems in our aging society. Strategies to prevent or delay the accumulation of the protein aggregates associated with these diseases have been recently proposed, raising significant hope; but currently, there is no perspective cure or preventive treatment for the damage to the brain [1]. Neurodegenerative processes are initiated long before clinical symptoms become obvious and proceed for years in a slow and irreversible manner. Therefore, it is of paramount importance to diagnose NDs as early as possible and distinguish between different disorders, with complex and similar symptoms, which might require different treatments. Early diagnosis is also critical for enrollment in clinical trials concerning new therapeutics, before patient exhibit mild or www.expert-reviews.com
10.1586/14737159.2013.849574
moderate dementia, to identify and apply drugs with the best chance of preserving cognitive functions. The ability to identify subjects at high risk for incipient dementia, long before the onset of cognitive deficit, represents a major advance in the field of neurodegenerative dementia [2]. AD is a progressive and fatal neurodegeneration, which is clinically characterized by progressive cognitive decline and inevitably leads to complete dependency on others for care. AD is the most common cause of dementia in the elderly [3], projected to affect 100 million people worldwide by 2050 [4]. Although a definitive diagnosis of AD is not possible until the detailed postmortem microscopic examination of the brain, AD is currently diagnosed using a combination of tools, such as the assessment of cognitive functions through neuropsychological tests and the study of the patient’s family and brain imaging through positron emission tomography or magnetic resonance imaging [3]. Unfortunately, the accuracy of the current clinical AD diagnostic methods is generally low [3]. However, new and established neurochemical biomarkers are increasingly applied to support clinical diagnosis, to aid in the design and evaluation of clinical trials, eventually leading to patient care. Progress in research related to biomarkers and
2013 Informa UK Ltd
ISSN 1473-7159
863
Review
Cretich, Sola, Gagni & Chiari
Vascular abnormality
Neuron Signalling molecule
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Aβ oligomers
Mitochondrion
Microglial cell
Nucleus
Truncated apoE4 Impaired synapse Aβ-degrading enzyme Tau
α-synuclein
Amyloid plaque ApoE4
Figure 1. The roles of key proteins in neurodegenerative disorders. The accumulation of b-amyloid in the brain (resulting from increased neuronal production, decreased degradation or altered transport) produces aggregation and amyloid plaques in the brain, thus impairing synaptic functions. The proteins tau and a-synuclein self-assemble into aggregates inside neurons thereby displacing vital organelles. Reproduced with permission from [4] Macmillan Publishers Ltd (2009).
the pathological changes associated with the progression of AD has resulted in new criteria and guidelines for AD in research settings [5], which incorporate biomarkers and a formal definition of the three phases of disease progression: preclinical AD, mild cognitive impairment (MCI) and AD-related dementia [2]. Each disease stage might be characterized using a specific biomarker profile (signature); moreover, the incorporation of biomarkers into clinical studies might speed up clinical trials. To date, three biomarkers in cerebrospinal fluid (CSF) have been well established and validated internationally to diagnose AD: b-amyloid (1–42) (Ab42), total tau protein and phosphorylated tau-181 [6]. The role of these proteins is showed in FIGURE 1, schematizing some of the key physiological players involved in the pathogenesis of AD. The accumulation and aggregation of b-amyloid in the brain might result from increased neuronal production, decreased degradation or altered transport functions. The proteins tau and a-synuclein assemble into aggregates inside neurons thereby displacing vital organelles. Physiologically, tau proteins interact with tubulin to stabilize microtubules. The hyperphosphorylation of tau proteins leads to the self-aggregation of the tangles inside neurons involved in AD and other neurodegenerative diseases. 864
However, the accuracy of biomarker measurement widely varies between studies [3]. Additional biomarkers will increase diagnostic accuracy. Novel biomarkers are also needed for the differential diagnosis of NDs and to identify mixed pathologies. Importantly, even if CSF is the most logical source of neurochemical markers, as it directly reflects metabolic processes in the brain, lumbar puncture is an invasive and delicate procedure, which cannot be used for screening and is restricted to patients with significant symptoms. Thus, it is crucial to discover and validate novel biomarkers in easily collectible samples such as plasma/serum. Basic and clinical research on NDs requires high-throughput methods to measure the profiles for both the increased acceptance of established biomarkers (through validation in clinical studies) and for new biomarker discovery. Ideally, the analytical tools for this purpose will provide at least three characteristics: sensitivity, multiplexing and high throughput. High sensitivity is a prerequisite, as low Ab42 levels in CSF are detected in preclinical disease stages and predict future cognitive decline and neurodegeneration, particularly an Ab42 concentration of
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Novel fluorescent microarray platforms: a case study in neurodegenerative disorders Expert Rev. Mol. Diagn. 13(8), 863–873 (2013)
Marina Cretich*, Laura Sola, Paola Gagni and Marcella Chiari Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy *Author for correspondence: Fax: +39 022 850 1239 [email protected]
This review focuses on the fluorescent protein microarrays applied to neurodegenerative disorders, a major health problem in our aging society. Biomarker discovery studies and work on new diagnostic tests are both included. Three platforms are described: antibody planar microarrays, comprising an array of well-defined antibodies for the simultaneous capture of biomarkers; antigen planar microarrays in which proteins, peptides or small molecules are immobilized and probed with biological samples to chase specific antibodies; and bead-based array platforms, which are flow cytometry-based methods in which capture molecules are coupled to fluorescent microspheres, facilitating the simultaneous quantification of several analytes. KEYWORDS: Alzheimer’s disease • antibody • biomarkers • fluorescence • microarrays • neurodegeneration
Neurodegenerative disorders (NDs) are characterized by a progressive loss of function of specific groups of neurons in different regions of the brain. The major disease groups are Alzheimer’s disease (AD), vascular dementia, Parkinson’s disease (PD), frontotemporal lobar degeneration, Lewy body dementia and amyotrophic lateral sclerosis. These disorders are variably associated with dementia, personality changes, language abnormalities or progressive muscle weakness. Because the greatest risk factor for NDs is age, these diseases are considered major health and quality of life problems in our aging society. Strategies to prevent or delay the accumulation of the protein aggregates associated with these diseases have been recently proposed, raising significant hope; but currently, there is no perspective cure or preventive treatment for the damage to the brain [1]. Neurodegenerative processes are initiated long before clinical symptoms become obvious and proceed for years in a slow and irreversible manner. Therefore, it is of paramount importance to diagnose NDs as early as possible and distinguish between different disorders, with complex and similar symptoms, which might require different treatments. Early diagnosis is also critical for enrollment in clinical trials concerning new therapeutics, before patient exhibit mild or www.expert-reviews.com
10.1586/14737159.2013.849574
moderate dementia, to identify and apply drugs with the best chance of preserving cognitive functions. The ability to identify subjects at high risk for incipient dementia, long before the onset of cognitive deficit, represents a major advance in the field of neurodegenerative dementia [2]. AD is a progressive and fatal neurodegeneration, which is clinically characterized by progressive cognitive decline and inevitably leads to complete dependency on others for care. AD is the most common cause of dementia in the elderly [3], projected to affect 100 million people worldwide by 2050 [4]. Although a definitive diagnosis of AD is not possible until the detailed postmortem microscopic examination of the brain, AD is currently diagnosed using a combination of tools, such as the assessment of cognitive functions through neuropsychological tests and the study of the patient’s family and brain imaging through positron emission tomography or magnetic resonance imaging [3]. Unfortunately, the accuracy of the current clinical AD diagnostic methods is generally low [3]. However, new and established neurochemical biomarkers are increasingly applied to support clinical diagnosis, to aid in the design and evaluation of clinical trials, eventually leading to patient care. Progress in research related to biomarkers and
2013 Informa UK Ltd
ISSN 1473-7159
863
Review
Cretich, Sola, Gagni & Chiari
Vascular abnormality
Neuron Signalling molecule
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.
Aβ oligomers
Mitochondrion
Microglial cell
Nucleus
Truncated apoE4 Impaired synapse Aβ-degrading enzyme Tau
α-synuclein
Amyloid plaque ApoE4
Figure 1. The roles of key proteins in neurodegenerative disorders. The accumulation of b-amyloid in the brain (resulting from increased neuronal production, decreased degradation or altered transport) produces aggregation and amyloid plaques in the brain, thus impairing synaptic functions. The proteins tau and a-synuclein self-assemble into aggregates inside neurons thereby displacing vital organelles. Reproduced with permission from [4] Macmillan Publishers Ltd (2009).
the pathological changes associated with the progression of AD has resulted in new criteria and guidelines for AD in research settings [5], which incorporate biomarkers and a formal definition of the three phases of disease progression: preclinical AD, mild cognitive impairment (MCI) and AD-related dementia [2]. Each disease stage might be characterized using a specific biomarker profile (signature); moreover, the incorporation of biomarkers into clinical studies might speed up clinical trials. To date, three biomarkers in cerebrospinal fluid (CSF) have been well established and validated internationally to diagnose AD: b-amyloid (1–42) (Ab42), total tau protein and phosphorylated tau-181 [6]. The role of these proteins is showed in FIGURE 1, schematizing some of the key physiological players involved in the pathogenesis of AD. The accumulation and aggregation of b-amyloid in the brain might result from increased neuronal production, decreased degradation or altered transport functions. The proteins tau and a-synuclein assemble into aggregates inside neurons thereby displacing vital organelles. Physiologically, tau proteins interact with tubulin to stabilize microtubules. The hyperphosphorylation of tau proteins leads to the self-aggregation of the tangles inside neurons involved in AD and other neurodegenerative diseases. 864
However, the accuracy of biomarker measurement widely varies between studies [3]. Additional biomarkers will increase diagnostic accuracy. Novel biomarkers are also needed for the differential diagnosis of NDs and to identify mixed pathologies. Importantly, even if CSF is the most logical source of neurochemical markers, as it directly reflects metabolic processes in the brain, lumbar puncture is an invasive and delicate procedure, which cannot be used for screening and is restricted to patients with significant symptoms. Thus, it is crucial to discover and validate novel biomarkers in easily collectible samples such as plasma/serum. Basic and clinical research on NDs requires high-throughput methods to measure the profiles for both the increased acceptance of established biomarkers (through validation in clinical studies) and for new biomarker discovery. Ideally, the analytical tools for this purpose will provide at least three characteristics: sensitivity, multiplexing and high throughput. High sensitivity is a prerequisite, as low Ab42 levels in CSF are detected in preclinical disease stages and predict future cognitive decline and neurodegeneration, particularly an Ab42 concentration of
