Identification of novel brain biomarkers.

BACKGROUND The diagnosis of diseases leading to brain injury, such as stroke, Alzheimer disease, and Parkinson disease, can often be problematic. In this study, we pursued the discovery of biomarkers that might be specific and sensitive to brain injury. METHODS We performed gene array analyses on a mouse model to look for biomarkers that are both preferentially and abundantly produced in the brain. Via bioinformatics databases, we identified the human homologs of genes that appeared abundant in brain but not in other tissues. We then confirmed protein production of the genes via Western blot of various tissue homogenates and assayed for one of the markers, visinin-like protein 1 (VLP-1), in plasma from patients after ischemic stroke. RESULTS Twenty-nine genes that were preferentially and abundantly expressed in the mouse brain were identified; of these 29 genes, 26 had human homologs. We focused on 17 of these genes and their protein products on the basis of their molecular characteristics, novelty, and/or availability of antibodies. Western blot showed strong signals in brain homogenates for 13 of these proteins. Tissue specificity was tested by Western blot on a human tissue array, and a sensitive and quantitative sandwich immunoassay was developed for the most abundant gene product observed in our search, VLP-1. VLP-1 was detected in plasma of patients after stroke and in cerebrospinal fluid of a rat model of stroke. CONCLUSIONS The use of relative mRNA production appears to be a valid method of identifying possible biomarkers of tissue injury. The tissue specificity suggested by gene expression was confirmed by Western blot. One of the biomarkers identified, VLP-1, was increased in a rat model of stroke and in plasma of patients after stroke. More extensive, prospective studies of the candidate biomarkers identified appear warranted.

[1]  W. Feinberg,et al.  Hemostatic markers in acute ischemic stroke. Association with stroke type, severity, and outcome. , 1996, Stroke.

[2]  M. Pelsers,et al.  Detection of brain injury by fatty acid-binding proteins , 2005, Clinical chemistry and laboratory medicine.

[3]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[4]  P. Coriat,et al.  Influence of hemolysis on the measurement of S-100beta protein and neuron-specific enolase plasma concentrations during coronary artery bypass grafting. , 2000, Clinical chemistry.

[5]  H. Lehrach,et al.  Analysis of the mouse proteome. (I) Brain proteins: Separation by two‐dimensional electrophoresis and identification by mass spectrometry and genetic variation , 1999, Electrophoresis.

[6]  J. Serena,et al.  Plasma Metalloproteinase-9 Concentration Predicts Hemorrhagic Transformation in Acute Ischemic Stroke , 2003, Stroke.

[7]  R. Anderson,et al.  Increase in serum S100A1-B and S100BB during cardiac surgery arises from extracerebral sources. , 2001, The Annals of thoracic surgery.

[8]  K. Blennow,et al.  Cerebrospinal fluid markers for Alzheimer's disease evaluated after acute ischemic stroke. , 2000, Journal of Alzheimer's disease : JAD.

[9]  T. Neumann-Haefelin,et al.  Predicts a Malignant Course of Infarction in Patients With Acute Middle Cerebral Artery Occlusion , 2004 .

[10]  L. Stead,et al.  Neuron-Specific Enolase as a Marker for Acute Ischemic Stroke: A Systematic Review , 2004, Cerebrovascular Diseases.

[11]  D. Laskowitz,et al.  Early biomarkers of stroke. , 2003, Clinical chemistry.

[12]  W. White,et al.  Novel Diagnostic Test for Acute Stroke , 2003, Stroke.

[13]  T. Neumann-Haefelin,et al.  Serum S100B Predicts a Malignant Course of Infarction in Patients With Acute Middle Cerebral Artery Occlusion , 2004, Stroke.

[14]  S. P. Fodor,et al.  High density synthetic oligonucleotide arrays , 1999, Nature Genetics.

[15]  H. Hårdemark,et al.  S-100 protein and neuron-specific enolase in cerebrospinal fluid and serum: markers of cell damage in human central nervous system. , 1987, Stroke.

[16]  J. Castillo,et al.  New Frontiers in the Diagnosis of Ischemic Stroke , 2003 .

[17]  P. Weinstein,et al.  Reversible middle cerebral artery occlusion without craniectomy in rats. , 1989, Stroke.

[18]  S. Anant,et al.  Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization. , 2003, Protein expression and purification.

[19]  H. Hampel,et al.  Blood‐Cerebrospinal Fluid Barrier Dysfunction for High Molecular Weight Proteins in Alzheimer Disease and Major Depression: Indication for Disease Subsets , 1997, Alzheimer disease and associated disorders.

[20]  J. Arenillas,et al.  Matrix Metalloproteinase Expression After Human Cardioembolic Stroke: Temporal Profile and Relation to Neurological Impairment , 2001, Stroke.

[21]  D. Seligson,et al.  Clinical Chemistry , 1965, Bulletin de la Societe de chimie biologique.

[22]  V. Arolt,et al.  S100B in brain damage and neurodegeneration , 2003, Microscopy research and technique.

[23]  Gert Lubec,et al.  Proteomics in brain research: potentials and limitations , 2003, Progress in Neurobiology.

[24]  F. Goodwin,et al.  Brain endolases as specific markers of neuronal and glial cells. , 1978, Science.

[25]  D. Lockhart,et al.  Expression monitoring by hybridization to high-density oligonucleotide arrays , 1996, Nature Biotechnology.

[26]  Melanie Hilario,et al.  Mining mass spectra for diagnosis and biomarker discovery of cerebral accidents , 2004, Proteomics.

[27]  Cornford,et al.  Blood-brain barrier permeability to small and large molecules. , 1999, Advanced drug delivery reviews.

[28]  R. Anderson,et al.  High serum S100B levels for trauma patients without head injuries. , 2001, Neurosurgery.

[29]  Peter Moyer,et al.  Recommendations for the Establishment of Stroke Systems of Care: Recommendations From the American Stroke Association’s Task Force on the Development of Stroke Systems , 2005, Circulation.

[30]  B. G. Blijenberg,et al.  Technical Performance and Diagnostic Utility of the New Elecsys® Neuron-Specific Enolase Enzyme Immunoassay , 2003, Clinical chemistry and laboratory medicine.

[31]  J. Brady,et al.  Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction , 1998, Nature.

[32]  A. Friedman,et al.  Serum von Willebrand Factor, Matrix Metalloproteinase-9, and Vascular Endothelial Growth Factor Levels Predict the Onset of Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage , 2002, Neurosurgery.

[33]  J S Alpert,et al.  Myocardial infarction redefined--a consensus document of The Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. , 2000, Journal of the American College of Cardiology.

[34]  W. Wisden,et al.  Expression of the neuronal calcium sensor protein family in the rat brain , 2000, Neuroscience.

[35]  Manfred Herrmann,et al.  Release of Glial Tissue–Specific Proteins After Acute Stroke: A Comparative Analysis of Serum Concentrations of Protein S-100B and Glial Fibrillary Acidic Protein , 2000, Stroke.

[36]  J. Vaage,et al.  Biochemical markers of neurologic injury in cardiac surgery: the rise and fall of S100beta. , 2001, The Journal of thoracic and cardiovascular surgery.

[37]  E. Fon,et al.  Hemostatic Markers in Acute Transient Ischemic Attacks , 1994, Stroke.

[38]  P. Lescuyer,et al.  Identification of post‐mortem cerebrospinal fluid proteins as potential biomarkers of ischemia and neurodegeneration , 2004, Proteomics.

[39]  S. Traynelis,et al.  Serine proteases and brain damage – is there a link? , 2000, Trends in Neurosciences.