biomarkers of disorders of the nervous system

Chapter 7Biomarkers of Disorders of the Nervous SystemIntroductionIn spite of all the advances in neurology, particularly in the last decade of thetwentieth century (Decade of the Brain), there are serious deciencies in our under-standing of the pathomechanism of several neurological disorders as well as ourability to diagnose and treat these disorders. Novel biomarker identication forneurological disorders will address the current shortcomings in their diagnosis andtherapeutics. Basic challenges to biomarker identication in neurological diseaseare the following: Limited availability of tissue from the site of pathology. Paucity of biomarkers of neurological disorders in blood, urine, and saliva. Access to CSF, the main source of biomarkers in CNS disorders, requires alumbar puncture. Poor clinical diagnostics and extent of disease progression at the time ofdiagnosis. The complexity of the brain and tissue heterogeneity. The lack of functional end points and models for validation.Discovery of Biomarkers of Neurological DisordersFigure 7.1 shows discovery and application of biomarkers in neurological diseases.Desirable characteristics of a biomarker vary according to the disease. Generalcharacteristics of an ideal biomarker of a CNS disease are as follows: It should be non-invasively (or minimally invasively) detectable in livingsubjects. Results should be reproducible. It should be positively correlated to the cause or the progression of the disease.K.K. Jain, The Handbook of Biomarkers, DOI 10.1007/978-1-60761-685-6_7,C Springer Science+Business Media, LLC 2010327328CNS tissueGenomicsDNAGenotypingmicroarrayRNALaser capturemicrodissection7 Biomarkers of Disorders of the Nervous SystemBloodCerebrospinal fluidMetabolomicsProteomicsSeparate proteinsSeparation ofmetabolitesIdentify proteinsAmplify RNASequencinggenes Expressionprofile Quantify proteins Identificationof metabolitesFunctional validation ofbiomarkers in modelsClinical studiesTherapeuticsValidation ofbiomarkersDiagnosticsValidation ofbiomarkersMonitoringtherapyFig. 7.1 Discovery and application of biomarkers in neurological diseasesPlasma or serum is the most convenient source of biomarkers. However, a patho-logical process in the CNS is not always reected in the systemic compartments, andthe detection of such biomarkers has been mostly limited to neurological diseasesthat have an autoimmune or a metabolic basis. Rened proteomic technologies arenow being used to detect biomarkers of neurodegenerative disorders in the blood.Biomarker Identication in the CSF Using ProteomicsCerebrospinal uid (CSF) is an important source of potential biomarkers of neu-rological disorders. CSF is also a rich source of biomarkers of systemic disorderssuch as peptides and antibodies that are capable of crossing the bloodbrain barrier(BBB). Proteomic technologies such as immunoblotting, isoelectric focusing, 2DGE, and MS have proven useful for deciphering the SCF proteome. CSF proteinsare generally less abundant than their corresponding serum counterparts, necessitat-ing the development and use of sensitive analytical techniques. Brain extracellularuid (ECF), mostly obtained by cerebral microdialysis and subjected to proteomicanalysis along with CSF, is also a good source of biomarkers of CNS disorders(Maurer 2009).Discovery of Biomarkers of Neurological Disorders 329A tandem mass tag approach, consisting of a set of structurally identical tags,which label peptides on free amino terminus and epsilon-amino functions of lysineresidues, has been used for quantitative MS-based proteomics of CSF (Dayon et al.2008). Human postmortem CSF was taken as a model of massive brain injury andcomparison was carried out with antemortem CSF. Peptides were identied andquantied by MS/MS analysis with MALDI-TOF/TOF and ESI-Q-TOF. The con-centration of 78 identied proteins was shown to be clearly increased in postmortemCSF samples compared to antemortem samples. Some of these proteins, like GFAP,protein S100B, and PARK7, are already known as biomarkers of brain damage, sup-porting the use of postmortem CSF as a valid model of brain insult. ELISA for theseproteins conrmed their elevated concentration in postmortem CSF.Biomarker Identication in the CSF Using LipidomicsLipids comprise the bulk of the dry mass of the brain. In addition to providingstructural integrity to membranes, insulation to cells and acting as a source ofenergy, lipids can be rapidly converted to mediators of inammation or to sig-naling molecules that control molecular and cellular events in the brain. ESI andatmospheric pressure chemical ionization have enabled compositional studies of thediverse lipid structures that are present in the brain. These include phospholipids,ceramides, sphingomyelin, cerebrosides, cholesterol, and their oxidized derivatives.Lipid analyses have delineated metabolic defects in neurological disorders. In thisreview, we examine the structure of the major lipid classes in the brain, describemethods used for their characterization, and evaluate their role in neurological dis-eases. Protein biomarkers in the CSF may be potential therapeutic targets sincethey transport lipids required for neuronal growth or convert lipids into moleculesthat control brain physiology. Combining lipidomics and proteomics will enhanceexisting knowledge of disease pathology and increase the likelihood of discoveringspecic markers and biochemical mechanisms of brain diseases.Cerebral Microdialysis for the Study of Biomarkers of CerebralMetabolismIn the past, the neurochemistry of the brain was primarily evaluated indirectly viasamples of the CSF, measurements of cerebral metabolism, or the evaluation ofspectra from nuclear magnetic resonance studies. In order to get direct estimatesof the neurochemical concentrations of the tissue, brain samples had to be col-lected and assayed a rather impractical approach for in vivo studies. Microdialysisoffered a unique opportunity to explore human brain neurochemistry without theneed for tissue extraction. Neurochemical biomarkers of cerebral metabolism thatcan be monitored by cerebral microdialysis are shown in Table 7.1.330 7 Biomarkers of Disorders of the Nervous SystemTable 7.1 Biomarkers of cerebral metabolismNeurochemical processDisturbed glucose metabolismExcitotoxicityIncreased adenosine triphosphate (ATP)utilizationATP depletionCellular membrane degradationReactive oxygen species formationNitric oxide formationNeurotransmitter releaseIonic perturbationsNeuroinammationBloodbrain barrier leakageBiomarkersGlucose, lactate, pyruvate,lactate/pyruvate, lactate/glucose, pHGlutamate (aspartate)Adenosine, inosine, hypoxanthineK+, neurotransmitter releaseGlycerolXanthine, urate, allantoin, ascorbate,glutathione, cysteine, spin-trapmetabolitesNitrite, nitrate, citrulline/arginine -Amino butyric acid, glycine,noradrenaline, dopamine, serotonin,adenosineNa+, Ca2+, Mg2+IL-1, IL-6, GFAP, NGFAlanine, valine, leucine Jain PharmaBiotech.Detection of Protein Biomarkers of CNS Disorders in the BloodAntibody-based tests can measure proteins in the blood. Various biomarkers foundin blood include S100 protein, neuron-specic enolase, myelin basic protein, andC-tau. These will be described under various neurological disorders later in thischapter.Concentrations of the S100 protein, an acidic calcium-binding protein found inthe gray matter of the brain, are elevated in serum after brain damage. Several com-mercial ELISA assays are available for S100 protein and are useful biochemicalmarkers for the early assessment of brain damage by the quantitative determinationof S100 in serum.Brain Imaging for Detection of BiomarkersImaging techniques enable the diagnosis of disease in vivo. Several specialized tech-niques have evolved during the past quarter of a century for imaging pathology in theliving brain. These include CT, MRI, PET, and SPECT (see Chapter 2). Althoughthese are used in diagnostic workup of neurological patients, the use in clinical trialshas been limited. Changes in brain pathology, visualized by imaging, can be used asan in vivo guide to the treatment. Brain imaging has an important role to play in thedetection of biomarkers of neurodegenerative diseases and these will be describedlater in this chapter.Biomarkers of Neural Regeneration 331Data Mining for Biomarkers of Neurological DisordersThe Stanley Medical Research Institute (/) onlinegenomics database (SMRIDB) is a comprehensive data mining tool to enableresearchers to elucidate the biological mechanisms of bipolar disorder, schizophre-nia, and depression. A diverse patient population combined with data generatedacross six microarray platforms and 12 studies to provide robust results to enhancethe understanding of brain disease (Higgs et al. 2006). It offers researchers anefcient tool for data mining of brain disease complete with information such ascross-platform comparisons and biomarkers elucidation for target discovery.Weighted correlation network analysis (WGCNA) can be used for nd-ing clusters (modules) of highly correlated genes (Langfelder and Horvath2008). Correlation networks facilitate network-based gene screening meth-ods that can be used to identify candidate biomarkers or therapeutic targets.The WGCNA package is freely available at /labs/horvath/CoexpressionNetwork/Rpackages/WGCNA.Antibodies as Biomarkers in Disorders of the Nervous SystemThe autoantibodies are routinely tested in many neuropathies, neuromuscular junc-tion disorders, and myopathies and can be very useful biomarkers for diagnosisand management. However, in CNS disorders, the pathogenic role of autoantibod-ies is still emerging. There is growing evidence for predominant antibody-mediateddemyelination in a subgroup of multiple sclerosis patients who may possibly beidentied by presence of autoantibodies. This will be discussed in more detaillater in this chapter. The autoantibodies in the peripheral nervous system can helpdetect a specic tumor and are routine tested in suspected cases of paraneoplasticsyndromes. The early detection and treatment of underlying tumor in peripheral ner-vous system may lead to clinical improvement in some cases. Clinical response toplasma exchange and intravenous immunoglobulin in certain CNS disorders associ-ated with anti-glutamic acid decarboxylase, GluR3, voltage-gated calcium channel,and voltage-gated potassium channel antibodies provides indirect evidence for apathogenic role of these autoantibodies. The search for more autoantibodies andthe effort to better dene their contribution to the disease process are ongoing. Inthe future, new paradigms for their detection and treatment of antibody-mediateddiseases may be established.Biomarkers of Neural RegenerationBiomarkers are useful for assessment of regeneration in the nervous system andalso for assessment of effect of agents such as neurotrophic factors such as nervegrowth factors (NGFs) that promote regeneration. One way to do this is to measure332 7 Biomarkers of Disorders of the Nervous Systemneurite outgrowth, which is a slow and tedious process. There is little work done onthis important topic, although more information is available about biomarkers thatimpede regeneration such as soluble Nogo-1.Surrogate biomarkers for neurite outgrowth activity were identied by geneexpression analysis in SH-O10 cells, subclones of the human SH-SY5Y neuroblas-toma cell line, which have a much higher NGF-induced neurite outgrowth activity(Oe et al. 2006). Microarray analysis revealed seven genes where mRNA levels werechanged. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, wereconrmed by quantitative real-time RT-PCR. Decreases in CyclinB2 and BIRC5mRNA induced by FK506 or retinoic acid, both of which enhance NGF-inducedneurite outgrowth effects, correlated with their neurite outgrowth activities. Theyconcluded that decreasing levels of CyclinB2 and BIRC5 mRNA strongly corre-late with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Ysubclonal cells, and have potential to become quantitative surrogate biomarkers formeasuring NGF-related neurite outgrowth.Biomarkers of Disruption of BloodBrain BarrierBloodbrain barrier (BBB) is a dynamic conduit for transport between blood andbrain of those nutrients, peptides, proteins, or immune cells that have access tocertain transport systems localized within the BBB membranes. Recent advancesin cell and molecular biology have provided new insights into the function of theBBB. Several disorders of the CNS are associated with increased permeability ofthe BBB. Two main approaches are used for studying the integrity of human BBBin vivo: (1) structural imaging employs contrast agents that only penetrate the BBBat sites of damage and (2) functional imaging is used to study the transport of sub-stances across the BBB both intact and damaged. Structural imaging employscontrast agents with CT scanning and is relatively insensitive. MRI with the con-trast agent gadolinium is more sensitive. Functional imaging is done with PET andcan quantify cerebral uptake of therapeutic agents, such as cytotoxic agents andmonoclonal antibodies. SPECT is less versatile than PET but can provide semi-quantitative measurement of BBB leakage of albumin or red blood cells. There is aneed for biomarkers to detect early changes in BBB.Loss of integrity of the BBB resulting from ischemia/reperfusion is believed to bea precursor to hemorrhagic transformation (HT) and poor outcome in acute strokepatients. A novel MRI biomarker was used to characterize early BBB disruptionin human focal brain ischemia and its association with reperfusion, HT, and pooroutcome (Latour et al. 2004). Reperfusion was found to be the most powerful inde-pendent predictor of early BBB disruption and thus of HT and is important for thedecision on acute thrombolytic therapy. Early BBB disruption as dened by thisimaging biomarker is a promising target for adjunctive therapy to reduce the com-plications associated with thrombolytic therapy, broaden the therapeutic window,and improve clinical outcome in acute stroke.Biomarkers of Neurotoxicity 333The astrocytic protein S100B is a potentially useful peripheral marker of BBBpermeability. Other biomarkers of BBB have recently been discovered by proteomicapproaches. These proteins are virtually absent in normal blood, appear in serumfrom patients with cerebral lesions, and can be easily detected by commerciallyavailable ELISA tests. These will be mentioned later in this chapter.Biomarkers of NeurotoxicityNeurotoxicity may be dened as any adverse effect of a biological, a chemical, ora physical agent on the structure or the function of the central and/or the periph-eral nervous system. A multidisciplinary approach is necessary to assess adult anddevelopmental neurotoxicity due to the complex and diverse functions of the ner-vous system. The overall strategy for understanding developmental neurotoxicityis based on two assumptions: (1) signicant differences in the adult versus thedeveloping nervous system susceptibility to neurotoxicity exist and they are oftendevelopmental stage dependent; (2) a multidisciplinary approach using neurobio-logical, including gene expression assays, neurophysiological, neuropathological,and behavioral function is necessary for a precise assessment of neurotoxicity.Application of genomic approaches to developmental studies must use the samecriteria for evaluating microarray studies as those in adults including considerationof reproducibility, statistical analysis, homogenous cell populations, and conrma-tion with non-array methods. The following statements are supported by studiesusing amphetamine to induce neurotoxicity: (1) gene expression data can help deneneurotoxic mechanism(s); (2) gene expression changes can be useful biomarkers ofeffect; and (3) the site-selective nature of gene expression in the nervous system maymandate assessment of selective cell populations. Desirable features of biomarkersof neurotoxicity are the following:Indicate response to diverse types of insults affecting any region of the brain.Sensitive with low incidence of false negatives.Specic to the neurotoxic condition with low incidence of false positives.Simple to evaluate.Quantitative.Glial Fibrillary Acidic Protein as Biomarker of NeurotoxicityThe glial reaction, gliosis, represents a hallmark of all types of nervous systeminjury. Therefore biomarkers of gliosis can be applied for assessment of neurotox-icity. The astroglial protein glial brillary acidic protein (GFAP) can serve as onesuch biomarker of neurotoxicity in response to a panel of known neurotoxic agents.334 7 Biomarkers of Disorders of the Nervous SystemQualitative and quantitative analysis of GFAP has shown this biomarker to be a sen-sitive and specic indicator of the neurotoxicity. The implementation of GFAP andrelated glial biomarkers in neurotoxicity screens may serve as the basis for furtherdevelopment of molecular signatures predictive of adverse effects on the nervoussystem.Single-Stranded DNA as a Biomarker of Neuronal ApoptosisSingle-stranded DNA (ssDNA) is a biomarker of apoptosis and programmed celldeath, which appears prior to DNA fragmentation during delayed neuronal death.A study on the immunohistochemical distribution of ssDNA in the brain was con-ducted to investigate apoptotic neuronal damage with regard to the cause of deathin medicolegal autopsy cases (Michiue et al. 2008). Neuronal immunopositivity forssDNA was globally detected in the brain, independent of the age, the gender ofsubjects, and the postmortem interval, and depended on the cause of death. Higherpositivity was typically found in the pallidum for delayed brain injury death andfatal carbon monoxide intoxication, and in the cerebral cortex, pallidum, and sub-stantia nigra for drug intoxication. For mechanical asphyxiatio