Description

	• Neuroproteomics technologies have been applied to the study of the central nervous system to identify cell types and tissues contributing to the disease.
	• Proteins and their proteolytic fragments released from degenerating neurons into the cerebrospinal fluid are biomarkers of brain damage.
	• An insight into protein-based mechanisms of disease will provide more relevant targets for drug discovery for neurologic disorders.

The function of the brain might be defined in terms of the number, type, and location of multiprotein complexes. A comprehensive list of complexes could provide a basis for maps of interactions or functional connection between complexes. Ascertaining the function of the complexes in the brain will require a combination of integrated approaches. To claim that a complex is important in any specific function, it will be necessary to demonstrate that interference with many of its individual proteins (by genetic or pharmacological interventions) interferes with the overall common function of the complex. Proteomics tools offer new ways to analyze networks of proteins that control important neurobiological phenomena. Functional neuroproteomics, combined with bioinformatics, can be used to study the organization of protein networks and molecular structures that underlie physiological, anatomical, and behavioral processes.

Various neuroproteomics technologies have been applied to the study of the central nervous system and may identify cell types and tissues contributing to the disease phenotype. Because the proteome of a cell or tissue is not a simple reflection of its transcriptome, direct protein-based analysis is needed.

Methods used in proteomics. The classical approaches in proteomics involve separating proteins by 2-dimensional gels and identifying visualizing bands or spots. A modification of this technique is 2-D polyacrylamide gel electrophoresis. Protein can be detected by Western blot or fluorescence detection methods. The proteins are then analyzed and characterized using mass spectrometry and its modification, matrix-assisted laser desorption mass spectrometry. Other methods used include capillary electrophoresis, high-performance liquid chromatography, and electrospray ionization (14). The most comprehensive source of information on proteomics is found in protein sequence databases. Currently, 2-D gel electrophoresis patterns are scanned into a computer and then analyzed by computer algorithms that quantify the gel patterns according to whether they are obtained under normal or physiologically altered conditions.

Application of proteomics to study functions of the nervous system. Proteomics technologies have been applied to the study of neurotransmitters, the synapse, and neuronal receptors. A brief description of these is given here.

Synapse. A synapse consists of an axon terminus, the presynapse, the synaptic cleft containing extracellular matrix proteins as well as adhesion molecules, and the postsynaptic density as target structure for chemical signaling. The proteomes of the presynaptic and postsynaptic active zones control neurotransmitter release and perception. Technologies such as single-cell proteomics enable measurement of long-term potentiation, which is known to be cell- as well as synapse-specific, and could contribute to our understanding of synaptic plasticity on a molecular level. Research on proteomics of the synapse has identified more than 2000 synapse proteins, tens of thousands of phosphorylation sites, and transient and time-resolved information on protein-protein interactions and structures, which has significantly increased our knowledge of the molecular composition and functioning of the synapse (35). Progress made in neuroproteomics of the synaptosome has helped in understanding the pathological alterations in the synaptic proteome in neurodegenerative disorders (01).

Neurotransmitters. Capillary electrophoresis has been combined with highly sensitive micro-electrospray-tandem mass spectrometry to simultaneously detect classical small molecule neurotransmitters as well as neuropeptides from discrete regions of the brain. Endogenous glutamate, gamma-aminobutyric acid, acetylcholine, and dopamine as well as the neuropeptides methionine-enkephalin and substance P 1-7 could be detected in the striatum using only a minute amount of brain tissue. A disease-specific quantitative analysis of a specific neurotransmitter of interest may require stabilization by inactivation of the degrading enzymes present in the cerebrospinal fluid.

N-methyl-D-aspartate receptors. Of the approximately 100 neuronal receptors known in humans, NMDA is described as an example. Proteomics technologies, such as mass spectrometry, can be used to characterize multiprotein complexes of NMDA receptors, which are encoded by activity-dependent genes.

Neuroprotection. Neuroproteomics, systems biology, and bioinformatics are used to assess the entire neuronal proteome. Neuroproteomics aids in the understanding of molecular mechanisms of neurogenesis. Potential neuroprotective pharmacological strategies can be targeted at Rho and Rho kinases, which constitute key integral points in the pathway that is known to be disrupted in multiple neuropathologies, such as spinal cord injury and traumatic brain injury.

Regeneration and degeneration of the nervous system. Neuroproteomic technologies have been designed to uncover the mechanisms and molecules involved in neuronal regeneration and degeneration. Most proteins mediating regeneration are found to be either malfunctioning or reduced in degeneration.

Study of the blood-brain barrier. Proteomic technologies can be used to study neuropathology at the blood-brain barrier. Proteomics can also be applied to facilitate drug delivery across the blood-brain barrier by characterizing active efflux systems that can prevent drug access to the brain and by identifying new transporters that could be used as noninvasive drug delivery conduits.

Neuroproteomics of the targets for deep brain stimulation. The nucleus basalis of Meynert and substantia nigra are the targets of deep brain stimulation. Although degeneration in these areas underlies cognitive decline in some neurologic disorders, knowledge about proteomics of this area is scanty. A proteomic study of these areas has revealed numerous proteins involved in the regulation of synaptic transmission and neurotransmitter release, and many of these are secreted in CSF as well as in plasma (08).

Use of neuroproteomics for study of neurologic disorders. Accumulation of aberrant proteins leads to various neurodegenerative disorders. A proteomic study has shown that the clearance of amyloid β peptide and diverse leaked cytosolic proteins in extracellular space is facilitated by the receptor-mediated degradation pathway where misfolded extracellular proteins are selectively captured by the extracellular chaperone clusterin and undergo endocytosis via the cell surface heparan sulfate receptor (12). This protein quality control system for preserving extracellular proteostasis plays a role in preventing diseases associated with aberrant extracellular proteins.

Neuropathologies involving loss of neurons and disturbances of neurotransmission may result in disease-specific alterations of neuronal and cerebrospinal fluid proteins that are suitable for proteomics analysis. Proteomics analysis of human brain tissue has been done as an extension of the study of cerebrospinal fluid proteins. These studies are further facilitated by the availability of 2-D maps of brain-specific proteins. Examples of some tissue proteomics studies in neurologic disorders are as follows:

Down syndrome. Brain tissue has been studied in Down syndrome, in which the main features of neuropathology are deteriorated migration, axonal pathfinding, and wiring of the brain, but information on the underlying mechanisms is still limited. Proteomics techniques such as 2-D electrophoresis with MALDI have been used to detect differences in protein expression between control, Down syndrome, and Alzheimer disease brains. Significantly decreased mRNA levels of dihydropyrimidinase-2 in 4 brain regions of patients with Down syndrome, but not with Alzheimer disease, as compared to controls were detected. 2-D electrophoresis revealed variable expression of DRP-2 proteins, which showed a high heterogeneity. The decrease of mRNA DRP-2 steady state levels in Down syndrome, along with deteriorated protein expression of this repulsive guidance molecule of the semaphorin and collapsin family, may help to explain deranged migration and histogenesis of Down syndrome brain.

Alzheimer disease. Use of proteomic methods to study brain tissue in early Alzheimer disease has shown that oxidative stress, particularly lipid peroxidation, is an early event in the progression of Alzheimer disease.

The replacement of a single amino acid can destabilize native proteins and trigger the aggregation of amyloid fibers that characterize a range of lethal neurodegenerative diseases. Neurodegenerative diseases associated with the accumulation of amyloid in the brain, including Huntington disease and Alzheimer disease, share certain features, despite implication of different proteins in each disease. Neurodegenerative diseases resulting from expanded repeat sequences of glutamine residues are associated with the formation of protein aggregates in the cell nuclei of the affected neurons. Nucleation of the aggregates is the controlling step in the progression of these diseases. One potential therapy for neurodegenerative diseases would be to use computational methods to find proteins that stabilize the native structure and counter the destabilizing effect of the glutamine repeats.

Quantitative proteome analysis has been used to compare signals corresponding to individual proteins between postmortem brain tissues from persons with Alzheimer disease, and those from age-matched, nondemented control tissues. Such comparative proteome analysis has the potential to provide new insights into pathogenic mechanisms in the dementias.

It is necessary to integrate the large amount of data concerning proteins involved in the pathogenesis of Alzheimer disease into a coherent global view of the disease. This requires a proteomics strategy combined with bioinformatics.

Clinically useful biomarkers of disrupted amyloid beta homeostasis in Alzheimer disease have been identified. Novel assays are expected to be developed by the most effective combinations of different biomarkers.

Proteomics approaches are helping to uncover the key steps leading to neurofibrillary degeneration and, thus, to identification of therapeutic targets for Alzheimer disease. Characterization by structural proteomics of relevant phosphosites that contribute to neurodegeneration could provide novel biomarkers and drug targets for treatment of Alzheimer disease.

Protein S-nitrosation (SNO-protein) impacts many biological systems, but the identification of the resulting proteins has been technically challenging. A chemical proteomic strategy, SNOTRAP (SNO trapping by triaryl phosphine), enables improved identification of SNO-proteins by mass spectrometry and reveals that S-nitrosation is elevated during early stages of neurodegeneration, preceding cognitive decline (31). Changes in the SNO-proteome during early neurodegeneration are relevant for synapse function, metabolism, and pathology of Alzheimer disease.

Loss of smell is an early symptom of Alzheimer disease and neuropathological abnormalities have been detected in the olfactory bulb. Proteome analysis shows that besides modulation of tau and amyloid precursor proteins, there is an impairment of synaptic proteostasis during evolution of the disease. Mitochondrial imbalance is evidenced by a depletion of Prohibitin-2 (Phb2) protein levels and a specific decrease in the phosphorylated isoforms of Phb1 in intermediate and advanced stages of Alzheimer disease (16). Phb complex is a driver of neurodegeneration at the olfactory level and provides a missing link in the understanding of the biochemical basis of olfactory dysfunction in Alzheimer disease.

Parkinson disease. Neuroproteomics is an important tool for research in Parkinson disease. Intraneuronal aggregates of alfa-synuclein occur in Lewy bodies and Lewy neurites, the cytopathological hallmarks of Parkinson disease. Destabilization of the helically folded tetramer precedes alpha-synuclein misfolding and aggregation in Parkinson disease; therefore, small molecules that stabilize the physiological tetramer could reduce alfa-synuclein pathogenicity (03). Proteomic profiling of substantia nigra has revealed that the endothelial/basement membrane pathway is tightly connected with Lewy body formation and dopaminergic neuronal loss (26). The poly(A) RNA-binding proteins, including the ones relevant to other neurodegenerative disorders, have a strong inverse correlation with Lewy bodies and may reflect an alternative mechanism of nigral neurodegeneration.

Neuroproteomics can help in establishing disease-associated protein profiles and might enable presymptomatic diagnosis of Parkinson disease. It is difficult to clinically differentiate Parkinson disease and multiple system atrophy, especially at the early stages of disease. Protein misfolding cyclic amplification is a technique that is used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity as well as specificity and may enable the development of a biochemical assay to discriminate between Parkinson disease and multiple system atrophy (32).

Use of proteomic technologies to research Parkinson disease has led to the concept that it may be an acquired or genetically determined brain proteinopathy involving an abnormal processing of several, rather than individual, neuronal proteins. Neuroproteomics has an underestimated potential to offer new, unbiased, and highly sensitive strategies to study the biological and molecular mechanisms underlying the pathology of Parkinson disease and to identify diagnostic, prognostic, or therapeutic biomarkers.

Cytosolic nonspecific dipeptidase 2, a protein not yet reported to be associated with pathogenesis of Parkinson disease, was shown to be increased in the substantia nigra of these patients, and it plays a key role in neurodegeneration, by mechanisms that could involve oxidative stress, protein aggregation, or inflammation (18).

Amyotrophic lateral sclerosis. Proteomic analysis can identify protein biomarkers that provide an insight into disease pathogenesis and are useful for diagnosis. CSF levels of tau protein and neurofilaments, biomarkers of axonal damage, are significantly more elevated in patients with amyotrophic lateral sclerosis than in controls. In CSF specimens, mass spectrometry peaks for cystatin C and transthyretin are reduced in amyotrophic lateral sclerosis, whereas peaks for posttranslational modified transthyretin and C-reactive protein are increased, predicting amyotrophic lateral sclerosis with an overall accuracy of 82% (28). Measurement of galectin-3 in CSF samples showed that amyotrophic lateral sclerosis patients had approximately twice as much galectin-3 as normal and disease controls (36).

Central nervous system trauma. Proteomics technologies are being used for mapping changes in proteins after injury. Spatially resolved microproteomics guided by mass spectrometry imaging, sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction are accurate and precise neuroproteomic techniques for the study of traumatic brain injury (22). These technologies will be useful for developing diagnostic predictors after CNS injury and to identify new therapeutic targets. Following severe traumatic brain injury, the number of extracellular microvesicles/exosomes released into CSF is increased. These microvesicles/exosomes contain several known as well as previously undescribed protein biomarkers of traumatic brain injury, which may contain cell to cell communication factors related to both neuron apoptosis signaling as well as neurodegeneration pathways (23). Neuroproteomics provides a molecular approach to analyze the complex secondary events after traumatic brain injury. Neuroproteomic response in a rat model of mild-moderate controlled cortical impact within first 2 weeks after injury analyzed by use of artificial neural networks identified loss of potassium/chloride cotransporter 2 (KCC2), 70% of which could be restored by targeting it with a KCC2 modulator within a time window of 1 hour to 1 day, but not if given too early or too late (19). This approach supports the therapeutic promise of KCC2-targeted CLP290 intervention for positive functional recovery after traumatic brain injury.

Proteins 14-3-3 zeta and 14-3-3 beta are released in the cerebrospinal fluid following traumatic brain injury, indicating that these proteins and their proteolytic fragments released from degenerating neurons are cerebrospinal fluid biomarkers of acute brain damage. Large-scale neuroproteomics research in traumatic brain injury is being applied for immediate biomarker discovery, and the systems biology approach is used for understanding how the brain responds to trauma. Neuroproteomic studies in experimental animals have helped to reveal similarities as well as differences between traumatic brain injury and ischemic brain injury at the molecular level that underlie the pathobiology of these 2 types of injury. Eventually, the knowledge gained through neuroproteomics could lead to improvement in diagnostics and therapeutics of central nervous system trauma.

Stroke. Determination of time of onset of stroke is important for deciding initiation of thrombolytic therapy for stroke. Blood level of several proteins have been measured by immunoassays in stroke patients to find a clinically significant biomarker of stroke (33). Glutathione S-transferase-π was found to accurately predict the time of stroke onset in over 50% of early stroke patients, and this test could, therefore, complement current guidelines for tPA administration and potentially increase the number of patients accessing thrombolytic therapy.

Epilepsy. Neuroproteomic technologies, particularly mass spectrometry, are being applied to research on epilepsy. Areas of research include discovery of protein biomarkers of epilepsy and exploration of the epileptic hippocampus proteome. Proteomic analyses in epilepsy are useful because the analyzed tissue sample closely represents the pathogenic process involved in human epilepsies, but the lack of an ideal animal experimental model of human epilepsy makes it difficult to translate these findings, and use of tissue samples from patients is also problematic because of individual variability (05). Eventually, advances in neuroproteomics could lead to improvements in understanding the pathogenesis, diagnosis, and treatment of epilepsy.

Psychiatric disorders. Proteomic technologies have been used to survey postmortem tissue to identify changes linked to the various psychiatric diseases such as schizophrenia. Two-dimensional gel electrophoresis and mass spectrometric sequencing of proteins enable the comparison of subsets of expressed proteins among a several samples. Protein species that display disease-specific alterations in level in the frontal cortex include forms of glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, and ubiquinone cytochrome C reductase core protein 1. Proteomics analysis, thus, may identify novel pathogenic mechanisms of human neuropsychiatric diseases.

Application of proteomic technologies, particularly mass spectrometry, has the potential to improve the understanding of the biochemical basis of psychiatric disorders and enable discovery of biomarkers as a basis for the development of diagnostics and improved therapeutics. Further studies of neuroproteomics of schizophrenia (with focus on subproteomes), along with the use of blood-based biomarkers, has a huge potential to improve our understanding of schizophrenia on a cellular level (07). Mass spectrometry-based proteomics has been used to study corpus callosum, which is altered in schizophrenia patients. Among the differentially expressed proteins are 14-3-3 proteins and myelin basic protein, indicating that these proteins may be biomarkers of schizophrenia (29).

Drug abuse. Proteomic technologies can be used to identify key proteins involved in drug abuse behaviors, with the aim of understanding the cause of drug abuse and identifying targets for the development of therapeutic agents (20). Neuroproteomics helps to discover relevant biomarkers for early diagnosis of alcoholism and to identify future pharmacological targets for the treatment (10).

Brain tumors. Proteomics technologies have been used for identification of differentially expressed proteins in human glioblastoma cell lines and tumors. Proteins that are highly expressed in glioblastoma multiforme compared to nonmalignant brain tissues may be used as diagnostic biomarkers for glioblastoma tumors. The application of proteomics techniques on pediatric brain tumors and CSF has enabled the discovery of proteins that can be used as potential disease biomarkers for diagnosis as well as prognosis or as molecular targets for developing new therapies (02; 30).

Desorption electrospray ionization-mass spectrometry during surgery of glioblastoma provides direct information on tumor infiltration into white or gray brain matter within a few minutes based on N-acetylaspartate and on membrane-derived complex lipids (27). This technology has enabled estimation of high tumor cell percentage at surgical margins with 93% sensitivity and 83% specificity, which is more practical and accurate than intraoperative biopsies and postoperative MRI. This technique of analysis of tissue smears, ex vivo, can be inserted into the current workflow for surgery of brain tumors without any disruption.

Analysis of resected specimens of childhood ependymoma by 1-D nanoLiquid chromatography-mass spectrometry has revealed many similarities in proteome content with 2 other pediatric brain tumors--astrocytomas and medulloblastomas. Furthermore, most of the currently proposed biomarkers for ependymoma, eg, nucleolin, nestin, Ki67 and laminin subunit A2 as well as all major key players of the phosphoinositide 3-kinase pathway were definitely detected (34).

Mitochondrial disorders. Proteomics technologies can characterize mitochondrial proteins directly involved in diseases associated with or caused by mitochondrial dysfunction. Apart from the mitochondrial disorders involving the nervous system, oxidative stress in the mitochondria is associated with the pathogenesis of neurodegenerative disorders. Future research will link proteomics technologies to the genomic information.

Brain aging. Age-related impairment of function of the central nervous system is associated with increased protein oxidation, which is believed to contribute to age-related learning and memory deficits. Quantitative proteomics has been used to study regional brain tissues of human patients for temporal expression of the synaptic proteome throughout the adult lifespan in differentially vulnerable brain regions, which will not only help in understanding the molecular mechanisms of brain aging but will also help in deciphering the regulatory biochemical cascades governing neurodegenerative disease pathogenesis (11).

Prion disorders. The cellular form of prion protein PrP(C) is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrP(Sc) to cause neurodegenerative diseases. Protein misfolding is implied in prion diseases affecting the nervous system. Several studies have shown that the phenomenon of prion aggregation may have a wider physiological importance, which is not fully understood. It may involve maintaining neuronal functions and possibly contributing to the establishment of long-term memory (25). PrP(C), via protein kinase A signaling, plays an important role in regulating synaptic plasticity in the developing hippocampus (04).

Chronic pain. Identification of several small peptides in CSF of patients with chronic pain supports the concept that a central neuroimmune response is initiated, which leads to central sensitization (24). Aim of therapy is to modulate and inhibit this response. Proteomic studies go beyond single molecule analysis by identifying the components of biological networks and pathways in chronic pain and defining their interactions, which offers the potential to provide a molecular overview of the biological processes involved in chronic pain. This will also facilitate examination of gene-drug interactions. An understanding of the central biological responses that result in chronic pain may facilitate the development of better therapies for chronic pain.

Clinical applications

Cerebrospinal fluids tests based on proteomics. Cerebrospinal fluid examination provides a practical way to conduct longitudinal molecular analyses of changes during neurologic diseases. Integrated and parallel analyses of neurotransmitters, neuropeptides, and fingerprints of proteins in the cerebrospinal fluid may provide a better insight into underlying pathomechanisms. Proteomics investigations of cerebrospinal fluid have led to the discovery and validation of biomarkers, mainly in the field of neurodegenerative disorders (09). Some disease-specific proteins identified in the cerebrospinal fluid of patients with neurologic disorders are shown in Table 1.

Table 1. Disease-Specific Proteins in the Cerebrospinal Fluid of Patients with Selected Neurologic Disorders

Proteins	Diseases
Tau proteins	• Alzheimer disease • Parkinson disease • Creutzfeldt-Jakob disease • AIDS encephalopathy • Alcohol-induced organic brain disorders
14-3-3 protein	• Creutzfeldt-Jakob disease
Dopamine-releasing protein	• Parkinson disease
Neurofilament protein	• Multiple sclerosis • Progressive supranuclear palsy
Myelin basic protein	• Multiple sclerosis • Optic neuritis
Neuron-specific enolase	• Cerebral infarction • Temporal lobe epilepsy
S-100 protein	• Cerebral infarction • Traumatic central nervous system injury • Temporal lobe epilepsy
Glial fibrillary acidic protein	• Severe neurodegeneration
Apolipoprotein D	• Alzheimer disease • Traumatic brain injury
Apolipoprotein E	• Alzheimer disease
Synaptosomal-associated protein	• Alzheimer disease
Amyloid precursor proteins	• Alzheimer disease
Group box protein-1	• Subarachnoid hemorrhage

Tau protein. An ELISA method is used to measure tau protein in the cerebrospinal fluid. Overall, an increase in tau protein concentration in cerebrospinal fluid has been described in Alzheimer disease, but several issues remain unclear. Extensive and accurate analysis of cerebrospinal fluid could be helpful to define tau protein species present in physiological conditions or released during the progression of a given neurodegenerative disease. Thus, determining the isoform content of tau should add specificity to the biological test, as many neurodegenerative disorders can be distinguished by their set of tau isoforms that accumulate in neurons. No commercial test is yet available for this purpose.

Protein 14-3-3. Analysis of cerebrospinal fluid by 2-D gel electrophoresis reveals the presence of a protein, designated as 14-3-3, which can be used to discriminate between Creutzfeldt-Jakob disease and other types of dementia. In patients with dementia, a positive immunoassay for the 14-3-3 protein in cerebrospinal fluid strongly supports a diagnosis of Creutzfeldt-Jakob disease. This finding does not support the use of the test in patients without clinically evident dementia.

S-100 and neuron-specific enolase. Both of these are elevated in the cisternal cerebrospinal fluid in patients with intractable temporal lobe epilepsy.

Group box protein-1. This is a potential CSF protein biomarker of neurologic outcome following subarachnoid hemorrhage in humans (15).

Diagnosis of neurologic disorders by examination of proteins in urine. The AD7C test detects elevated levels of a brain protein, neural thread protein (NTP), in the urine of patients suspected of having Alzheimer disease. Overexpression of the AD7C-NTP gene is associated with cell death like that found in the brain in Alzheimer disease. AlzheimAlert, a competitive ELISA assay, has been tested in urine samples from patients with Alzheimer disease and healthy normal individuals. It was found to be an accurate method in prospective and retrospective double-blind controlled studies for determining AD7C-NTP levels to help physicians in the diagnosis of Alzheimer disease. This test kit is now certified with a CE Mark, making it eligible for sale in the European Union. Another urine AD7C-NTP ELISA kit has been described, which may be a useful diagnostic kit for detecting early Alzheimer disease in a Chinese population (21).

Diagnosis of neurologic disorders by examination of proteins in the blood.

S-100. Antibody-based tests can measure proteins in the blood. Concentrations of the S-100 protein, an acidic calcium-binding protein found in the gray matter of the brain, are elevated in serum after brain damage. Measurement of serum concentrations of S-100 is a valuable tool that can be used more easily than tests on cerebrospinal fluid in the differential diagnosis of Creutzfeldt-Jakob disease, as significantly higher concentrations are found in Creutzfeldt-Jacob disease than in other diseases. However, this test does not replace brain biopsy for definitive diagnosis of Creutzfeldt-Jakob disease.

Several commercial ELISA assays are available for S-100 protein and are useful biochemical markers for the early assessment of cerebral infarction by the quantitative determination of S-100 serum. Undetectable S-100 in the blood of patients with head injury can rule out brain damage, and S-100 levels correlate with the extent of brain damage in severe head injury. Peak levels of serum S-100 correlate with neurologic deficit resulting from either stroke or traumatic brain injury, and the patterns can be used to differentiate between the 2 conditions (06). Undetectable serum level of S-100 protein predicts normal intracranial findings on CT scan in patients with traumatic brain injury. Determination of S-100 protein in serum may be used to select patients for CT scanning.

Elevated serum levels of S-100 in patients with liver cirrhosis indicate early and subclinical portal-systemic encephalopathy. It seems to be a promising biochemical surrogate marker for mild cognitive impairments due to portal-systemic encephalopathy.

Neuron-specific enolase. This is a glycolytic enzyme found in the neurons and neuroendocrine cells. Increased levels of neuron-specific enolase due to ischemic stroke have been measured in cerebral spinal fluid as well as in blood. Small infarcts and transient ischemic attacks elevate only neuron-specific enolase. Peak levels of both neuron-specific enolase and S-100 are statistically significant and correlate with clinical measures of stroke size and outcome.

Myelin basic protein. This is localized in the myelin sheath and constitutes approximately one third of the total protein of myelin from the human brain. Various studies have indicated that myelin basic protein concentrations in plasma and serum can be used as biomarkers for brain damage and severity of stroke. The increase of myelin basic protein in cerebral infraction is most evident several days after the onset, whereas in cerebral hemorrhage, the peak increase occurs almost immediately after the onset.

Transthyretin. This is a marker protein that extravasates into the blood from the CSF only if there is disruption of the CSF-blood barrier. Transthyretin can be detected in the blood by proteomics technologies and is potentially useful for the diagnosis of diseases where the CSF-blood barrier is disrupted.

Role of proteomics in neuropharmacology. An insight into protein-based mechanisms of neurologic disorders will provide more relevant targets for drug discovery for central nervous system disorders. Neurodegenerative diseases with underlying protein abnormalities are shown in Table 2.

Table 2. Neurodegenerative Diseases with Underlying Protein Abnormalities

Disease	Proteins involved	Inclusion bodies
Familial encephalopathy with myoclonus	Neuroserpin	Collin body
Familial Parkinson disease with Lewy bodies	Alpha-synuclein	Lewy body
Creutzfeldt-Jakob disease	Prion protein	vCJD amyloid
Alzheimer disease	Beta-amyloid peptide	Beta-amyloid plaques
Pick disease	Tau protein	Pick body
Huntington disease	Soluble huntingtin	Insoluble huntingtin
GM1 gangliosidosis	Deficiency of beta-gal	GM1-ganglioside

Neuroproteomics studies show that neurodegenerative diseases share many common molecular mechanisms, including misfolding of proteins. Further understanding of these mechanisms may lead to strategies for prevention of neurodegenerative diseases. Identification of neurodegeneration-associated changes in protein expression will facilitate the identification of novel biomarkers for the early detection of neurodegenerative diseases.

Approximately half of all drug targets are key central nervous system membrane proteins. Application of neuroproteomics technologies, therefore, hold great promise for improvements in the understanding, diagnosis, and therapy of central nervous system disorders.

As the molecular mechanisms of action of some of the classical drugs on the brain are being identified, protein kinase C has been implicated in many disorders, such as depression. Valproic acid, used for the treatment of manic-depressive disorders, decreases protein kinase C, and this effect coincides with an increase in neuroprotective protein bcl-2 in the central nervous system. Chronic lithium administration for manic-depressive disorder also produces a reduction in the expression of protein kinase C alpha and epsilon. Further, studies have demonstrated robust effects of lithium on another kinase system, GSK-3beta, and on neuroprotective and neurotrophic proteins in the brain. These observations have implications for the design of new therapies for neurologic disorders.

Circumstantial evidence from the mouse and Drosophila model systems suggests that abnormal protein folding and aggregation play a key role in the pathogenesis of both Huntington disease and Parkinson disease. Therefore, a detailed understanding of the molecular mechanisms of protein aggregation and its effect on neuronal cell death could open new opportunities for therapy.

Future of neuroproteomics. Advances in sensitivity and speed of mass spectrometers has enabled quantitative assessment of thousands of proteins from tissues and is beginning to reveal the alteration of proteins and their networks during progression of neurodegeneration as well as identify candidate proteins as potential biomarkers for specific neurodegenerative diseases (17). Further increase in use of proteomics technologies will facilitate a more comprehensive analysis of novel therapeutic strategies for central nervous system disorders. Despite rapid advances in proteomic technologies, neuroproteomics faces special challenges because of the complex cellular and subcellular architecture of the central nervous system. There are further challenges in integrating neuroproteomic data with other data modalities to achieve finer cellular and subcellular resolution in studies of neural tissues. A central nervous system proteome database of primary human neural tissues may avoid uncertainties of experimental models and accelerate pre- and clinical development of more specific diagnostic and prognostic disease markers and new selective therapeutics.