Dynamics of extracellular matrix proteins in cerebrospinal fluid and serum and their relation to clinical outcome in human traumatic brain injury.

Background Brevican, neurocan, tenascin-C and tenascin-R are extracellular matrix proteins present in brain that show increased expression in experimental animal models of brain injury. However, little is known about the dynamics of these proteins in human body fluids, such as cerebrospinal fluid (CSF) and serum, after traumatic brain injury (TBI). The aims of this study were to investigate if matrix proteins in CSF and serum are associated with functional outcome following traumatic brain injury, if their concentrations change over time and to compare their levels between brain injured patients to controls. Methods In total, 42 traumatic brain injury patients, nine healthy controls and a contrast group consisting of 38 idiopathic normal pressure hydrocephalus patients were included. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of proteins. Results Increased concentrations of brevican, tenascin-C and tenascin-R in CSF correlated with unfavourable outcome, with stronger outcome prediction ability compared to other biomarkers of brain tissue injury. CSF brevican, tenascin-R and serum neurocan gradually decreased with time (p = 0.04, p = 0.008, p = 0.005, respectively), while serum tenascin-C (p = 0.01) increased. CSF concentrations of brevican, neurocan and tenascin-R (only in time point 3) after TBI were lower than in the idiopathic normal pressure hydrocephalus group (p < 0.0001, p < 0.0001, and p = 0.0008, respectively). In serum, tenascin-C concentration was higher and neurocan lower compared to healthy controls (p = 0.02 and p = 0.0009). Conclusions These findings indicate that levels of extracellular matrix proteins are associated with clinical outcome following TBI and may act as markers for different pathophysiology than currently used protein biomarkers.

In-Depth Cerebrospinal Fluid Quantitative Proteome and Deglycoproteome Analysis: Presenting a Comprehensive Picture of Pathways and Processes Affected by Multiple Sclerosis.

In the current study, we conducted a quantitative in-depth proteome and deglycoproteome analysis of cerebrospinal fluid (CSF) from relapsing-remitting multiple sclerosis (RRMS) and neurological controls using mass spectrometry and pathway analysis. More than 2000 proteins and 1700 deglycopeptides were quantified, with 484 proteins and 180 deglycopeptides significantly changed between pools of RRMS and pools of controls. Approximately 300 of the significantly changed proteins were assigned to various biological processes including inflammation, extracellular matrix organization, cell adhesion, immune response, and neuron development. Ninety-six significantly changed deglycopeptides mapped to proteins that were not found changed in the global protein study. In addition, four mapped to the proteins oligo-myelin glycoprotein and noelin, which were found oppositely changed in the global study. Both are ligands to the nogo receptor, and the glycosylation of these proteins appears to be affected by RRMS. Our study gives the most extensive overview of the RRMS affected processes observed from the CSF proteome to date, and the list of differential proteins will have great value for selection of biomarker candidates for further verification.

Evaluating EFEMP1 in Cerebrospinal Fluid and Serum as a Potential Diagnosis Biomarker for Meningiomas.

BACKGROUND: The aim of this study was to determine the cerebrospinal fluid (CSF) and serum levels of EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) in the patients with meningiomas and explore its potential as a biomarker. METHODS: Forty-five patients with meningioma, 11 of whom underwent meningioma resection, as well as 30 healthy controls were enrolled in this study. CSF and blood samples were collected preoperatively and postoperatively. Expression levels of EFEMP1 were measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves were used to evaluate its discriminant ability. RESULTS: CSF EFEMP1 levels were significantly higher in the CSF samples (p < 0.0001) and serum samples (p = 0.0056) of meningioma patients compared to controls. To distinguish meningioma patients from controls by CSF and serum EFEMP1 levels, ROC/AUC analysis indicated an AUC of 0.945 (sensitivity 0.933; specificity 0.833) and an AUC of 0.674 (sensitivity 0.867; specificity 0.400), respectively. Moreover, the postoperative CSF levels of EFEMP1 were significantly decreased compared to the preoperative levels (p < 0.0001). CONCLUSIONS: The present study suggested that elevated EFEMP1 levels might be a novel diagnostic biomarker for meningioma patients.

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery. METHODS: Paired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays. RESULTS: Brevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = - 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84-0.94) compared with those without. CONCLUSION: Levels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics.

Disease-Specific Changes in Reelin Protein and mRNA in Neurodegenerative Diseases.

Reelin is an extracellular glycoprotein that modulates neuronal function and synaptic plasticity in the adult brain. Decreased levels of Reelin activity have been postulated as a key factor during neurodegeneration in Alzheimer´s disease (AD) and in aging. Thus, changes in levels of full-length Reelin and Reelin fragments have been revealed in cerebrospinal fluid (CSF) and in post-mortem brains samples of AD patients with respect to non-AD patients. However, conflicting studies have reported decreased or unchanged levels of full-length Reelin in AD patients compared to control (nND) cases in post-mortem brains and CSF samples. In addition, a compelling analysis of Reelin levels in neurodegenerative diseases other than AD is missing. In this study, we analyzed brain levels of RELN mRNA and Reelin protein in post-mortem frontal cortex samples from different sporadic AD stages, Parkinson's disease with dementia (PDD), and Creutzfeldt-Jakob disease (sCJD), obtained from five different Biobanks. In addition, we measured Reelin protein levels in CSF samples of patients with mild cognitive impairment (MCI), dementia, or sCJD diagnosis and a group of neurologically healthy cases. The results indicate an increase in RELN mRNA in the frontal cortex of advanced stages of AD and in sCJD(I) compared to controls. This was not observed in PDD and early AD stages. However, Reelin protein levels in frontal cortex samples were unchanged between nND and advanced AD stages and PDD. Nevertheless, they decreased in the CSF of patients with dementia in comparison to those not suffering with dementia and patients with MCI. With respect to sCJD, there was a tendency to increase in brain samples in comparison to nND and to decrease in the CSF with respect to nND. In conclusion, Reelin levels in CSF cannot be considered as a diagnostic biomarker for AD or PDD. However, we feel that the CSF Reelin changes observed between MCI, patients with dementia, and sCJD might be helpful in generating a biomarker signature in prodromal studies of unidentified dementia and sCJD.

The diagnostic performance of a novel ELISA for human CTP (Cochlin-tomoprotein) to detect perilymph leakage.

Perilymphatic fistula is defined as an abnormal communication between the perilymph-filled space and the middle ear, or cranial spaces. The manifestations include a broad spectrum of neuro-otological symptoms such as hearing loss, vertigo/dizziness, disequilibrium, aural fullness, tinnitus, and cognitive dysfunction. By sealing the fistula, perilymphatic fistula is a surgically correctable disease. Also, appropriate recognition and treatment of perilymphatic fistula can improve a patient's condition and hence the quality of life. However, the difficulty in making a definitive diagnosis due to the lack of an appropriate biomarker to detect perilymph leakage has caused a long-standing debate regarding its management. We have reported a clinical test for the diagnosis of perilymphatic fistula by detecting a perilymph specific protein, Cochlin-tomoprotein, as a diagnostic marker using a western blot. The aim of this study is to establish an ELISA-based human Cochlin-tomoprotein detection test and to evaluate its diagnostic accuracy in clinical subjects. The results of ELISA showed good dilution reproducibility. The mean concentration was 49.7±9.4 of 10 perilymph samples. The ROC curve in differentiating the perilymph leakage condition from the normal middle ear was significant (P < 0.001) with an area under the curve (AUC) of 0.918 (95% CI 0.824-0.100). We defined the diagnostic criteria as follows: CTP<0.4 negative; 0.4≦CTP<0.8 intermediate; 0.8≦CTP(ng/ml) positive in the clinical usage of the hCTP ELISA, and sensitivity and specificity were 86.4% and 100%, respectively. We further tested the expression specificity of the Cochlin-tomoprotein by testing blood and CSF samples. The concentration was below the detection limit (0.2 ng/ml) in 38 of the 40 blood, and 14 of the 19 CSF samples. We report the accuracy of this test for the diagnosis of perilymphatic fistula. Using ELISA, we can improve the throughput of the test. Furthermore, it is useful for a large-scale study to characterize the clinical picture and delineate the management of this medical condition.

Brain, blood, cerebrospinal fluid, and serum biomarkers in schizophrenia.

Over the last decade, finding a reliable biomarker for the early detection of schizophrenia (Scz) has been a topic of interest. The main goal of the current review is to provide a comprehensive view of the brain, blood, cerebrospinal fluid (CSF), and serum biomarkers of Scz disease. Imaging studies have demonstrated that the volumes of the corpus callosum, thalamus, hippocampal formation, subiculum, parahippocampal gyrus, superior temporal gyrus, prefrontal and orbitofrontal cortices, and amygdala-hippocampal complex were reduced in patients diagnosed with Scz. It has been revealed that the levels of interleukin 1ß (IL-1ß), IL-6, IL-8, and TNF-α were increased in patients with Scz. Decreased mRNA levels of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), neurotrophin-3 (NT-3), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) genes have also been reported in Scz patients. Genes with known strong relationships with this disease include BDNF, catechol-O-methyltransferase (COMT), regulator of G-protein signaling 4 (RGS4), dystrobrevin-binding protein 1 (DTNBP1), neuregulin 1 (NRG1), Reelin (RELN), Selenium-binding protein 1 (SELENBP1), glutamic acid decarboxylase 67 (GAD 67), and disrupted in schizophrenia 1 (DISC1). The levels of dopamine, tyrosine hydroxylase (TH), serotonin or 5-hydroxytryptamine (5-HT) receptor 1A and B (5-HTR1A and 5-HTR1B), and 5-HT1B were significantly increased in Scz patients, while the levels of gamma-aminobutyric acid (GABA), 5-HT transporter (5-HTT), and 5-HT receptor 2A (5-HTR2A) were decreased. The increased levels of SELENBP1 and Glycogen synthase kinase 3 subunit α (GSK3α) genes in contrast with reduced levels of B-cell translocation gene 1 (BTG1), human leukocyte antigen DRB1 (HLA-DRB1), heterogeneous nuclear ribonucleoprotein A3 (HNRPA3), and serine/arginine-rich splicing factor 1 (SFRS1) genes have also been reported. This review covers various dysregulation of neurotransmitters and also highlights the strengths and weaknesses of studies attempting to identify candidate biomarkers.

Cartilage oligomeric matrix protein and hyaluronan levels in synovial fluid from horses with osteoarthritis of the tarsometatarsal joint compared to a control population.

REASONS FOR PERFORMING STUDY: Quantification of cartilage oligomeric matrix protein (COMP) levels within synovial fluid from the tarsometatarsal joint has not previously been reported and an effective synovial fluid marker would allow monitoring of disease progression and treatment. OBJECTIVES: To quantify levels of COMP and hyaluronan (HA) in synovial fluid from the tarsometatarsal joint, identify differences in levels from horses with osteoarthritis (OA) of the tarsometatarsal joint compared to a control population and to correlate levels with radiographic changes in horses with OA. METHODS: Synovial fluid was collected from the tarsometatarsal joint of 25 horses without hindlimb lameness (controls) and 25 lame horses, subjected to analgesia of the joint. COMP concentrations were measured using a homologous inhibition ELISA. Immunoblots of synovial fluid from 3 lame horses and 3 controls were performed to identify fragmentation of COMP. Hyaluronan (HA) concentration in synovial fluid was determined using a competition ELISA. Radiographs of the lame horses with OA were scored and correlated with levels of COMP and HA. RESULTS: Concentrations of COMP in OA of the tarsometatarsal joint were significantly lower than in the control samples. An additional fragment band of COMP (approximately 30 kDa) was identified on the immunoblots of the horses with OA and this fragment was not identified in controls. No significant difference was identified in the HA or HA:COMP ratio between lame and control horses. There was no correlation between levels of synovial fluid COMP and HA, and radiographic changes. CONCLUSIONS AND POTENTIAL RELEVANCE: Lowered levels of COMP in synovial fluid of tarsometatarsal joints correlates with the presence of osteoarthritis. However, a single value cannot be used to stage the disease process. Levels of HA may not be a useful marker for this disease. Decreased, rather than increased COMP levels, may reflect significant loss of cartilage in established osteoarthritis. A specific assay for the COMP fragment generated with osteoarthritis may allow the earlier detection of clinical cases.

Blood and CSF biomarkers in brain subcortical ischemic vascular disease: Involved pathways and clinical applicability.

Vascular dementia is the second most common type of dementia after Alzheimer's disease (AD). Subcortical ischemic vascular disease refers to a form of vascular cognitive impairment characterized by the presence of diffuse white matter hyperintensities (WMHs) and multiple lacunar infarcts. These neuroimaging findings are mainly caused by cerebral small-vessel disease (cSVD) and relate to aging and cognitive impairment, but they can also be silent and highly prevalent in otherwise healthy individuals. We aimed to review studies on blood and cerebrospinal fluid (CSF) markers related to the presence of WMHs and lacunar infarcts that have been conducted in the past in large population-based studies and in high-risk selected patients (such as those with vascular risk factors, vascular cognitive impairment, or AD). Relevant associations with the presence and progression of cSVD have been described in the blood for markers related to inflammatory processes, endothelial damage and coagulation/fibrinolysis processes, etc. Also, different combinations of CSF markers might help to differentiate between etiologic types of dementia. In the future, to translate these findings into clinical practice and use biomarkers to early diagnosis and monitoring vascular cognitive impairment would require the replication of candidate markers in large-scale, multicenter, and prospectively designed studies.

Reelin immunoreactivity in neuritic varicosities in the human hippocampal formation of non-demented subjects and Alzheimer's disease patients.

BACKGROUND: Reelin and its downstream signaling members are important modulators of actin and microtubule cytoskeleton dynamics, a fundamental prerequisite for proper neurodevelopment and adult neuronal functions. Reductions in Reelin levels have been suggested to contribute to Alzheimer's disease (AD) pathophysiology. We have previously reported an age-related reduction in Reelin levels and its accumulation in neuritic varicosities along the olfactory-limbic tracts, which correlated with cognitive impairments in aged mice. Here, we aimed to investigate whether a similar Reelin-associated neuropathology is observed in the aged human hippocampus and whether it correlated with dementia status. RESULTS: Our immunohistochemical stainings revealed the presence of N- and C-terminus-containing Reelin fragments in corpora amylacea (CAm), aging-associated spherical deposits. The density of these deposits was increased in the molecular layer of the subiculum of AD compared to non-demented individuals. Despite the limitation of a small sample size, our evaluation of several neuronal and glial markers indicates that the presence of Reelin in CAm might be related to aging-associated impairments in neuronal transport leading to accumulation of organelles and protein metabolites in neuritic varicosities, as previously suggested by the findings and discussions in rodents and primates. CONCLUSIONS: Our results indicate that aging- and disease-associated changes in Reelin levels and proteolytic processing might play a role in the formation of CAm by altering cytoskeletal dynamics. However, its presence may also be an indicator of a degenerative state of neuritic compartments.

Immunohistochemical analysis of cartilage-derived retinoic acid-sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA) in murine, canine, bovine and equine cerebrospinal tissues.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA), which appears abundantly in hypertrophic cartilage at the stage of endochondral ossification, is also detected in cerebrospinal fluid (CSF) following spinal cord injury. In this study, the localization of the CD-RAP/MIA molecule in normal tissues of the spine and brain obtained from mice, rats, dogs, cattle and horses was examined using immunohistochemistry with a specific antibody. The positive signals of CD-RAP/MIA were found at nerve cells in the spinal cords of all species and were especially strong at cerebellar Purkinje cells. The results suggested that CD-RAP/MIA included in normal cerebrospinal tissues could be a biomarker associated with tissue injuries, as the molecules might flow into the CSF.

Analysis of cartilage oligomeric matrix protein and matrix metalloproteinase-9 in cerebrospinal fluid of miniature dachshund with intervertebral disc herniation.

We evaluated whether the cerebrospinal fluid (CSF) concentration of cartilage oligomeric matrix protein (COMP) is related to disease severity, prognosis and matrix metalloproteinase (MMP)-9 activity of the CSF in miniature dachshund with intervertebral disc herniation. Samples were obtained from 23 patients and 6 normal dogs, and all patients received hemilaminectomy. Twenty dogs recovered successfully and 3 of 11 dogs without deep nociception had MMP-9 activity in the CSF and an unsuccessful outcome. The COMP levels from patients were significantly higher than those from normal dogs. MMP-9 activity and neurological severity were not related to the COMP levels. However, the COMP levels from 3 unsuccessful cases that had MMP-9 activity were significantly lower than those from all recovered cases and/or successful cases without deep nociception. Concerning severe cases, increased proteolytic activity might affect the COMP concentration and prognosis due to MMP-9 associated deleterious effects.

Identification of SPARC-like 1 protein as part of a biomarker panel for Alzheimer's disease in cerebrospinal fluid.

We have used proteomic fingerprinting to investigate diagnosis of Alzheimer's disease (AD). Samples of lumbar cerebrospinal fluid (CSF) from clinically-diagnosed AD cases (n = 33), age-matched controls (n = 20), and mild cognitive impairment (MCI) patients (n = 10) were used to obtain proteomic profiles, followed by bioinformatic analysis that generated a set of potential biomarkers in CSF samples that could discriminate AD cases from controls. The identity of the biomarker ions was determined using mass spectroscopy. The panel of seven peptide biomarker ions was able to discriminate AD patients from controls with a median accuracy of 95% (sensitivity 85%, specificity 97%). When this model was applied to an independent blind dataset from MCI patients, the intensity of signals was intermediate between the control and AD patients implying that these markers could potentially predict patients with early neurodegenerative disease. The panel were identified, in order of predictive ability, as SPARC-like 1 protein, fibrinogen alpha chain precursor, amyloid-ß, apolipoprotein E precursor, serum albumin precursor, keratin type I cytoskeletal 9, and tetranectin. The 7 ion ANN model was further validated using an independent cohort of samples, where the model was able to classify AD cases from controls with median accuracy of 84.5% (sensitivity 93.3%, specificity 75.7%). Validation by immunoassay was performed on the top three identified markers using the discovery samples and an independent sample cohort which was from postmortem confirmed AD patients (n = 17).

Cartilage oligomeric matrix protein in canine spinal cord appears in the cerebrospinal fluid associated with intervertebral disc herniation.

STUDY DESIGN: Cerebrospinal fluid (CSF) from dogs with intervertebral disc herniation (IVDH) were analyzed using a specific antibody against cartilage oligomeric matrix protein (COMP). Immunolocalization and genetic expression of COMP were also examined in the spinal cords of mouse, rat, and dog. OBJECTIVE: To investigate the hypothesis that COMP is present in CSF of dogs with IVDH, and the clinical relevance of COMP expression in CSF as a potential biomarker of spinal cord injury. SUMMARY OF BACKGROUND DATA: In dog IVDH, diagnostic imaging is useful for determining the spinal cord compression, but less useful for assessment of traumatic degeneration. Aggrecan, a well-known component of cartilage matrix, is increased in CSF from cases of neural damage and inflammation of the spinal cord. METHODS: CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 19 dogs with IVDH and 5 normal dogs were analyzed using inhibition enzyme-linked immunosorbent assay (ELISA) and immunoblotting with an antibody cross-reactive with dog COMP. Samples of normal spinal cord from mouse, rat, and dog were also prepared for immunohistochemistry and in situ hybridization. RESULTS: ELISA values were significantly higher for L-CSF than for C-CSF in dogs with IVDH, whereas there was no significant difference between them in normal dogs. Immunoblots of L-CSF samples revealed positive bands of approximately 500 kDa in 6 cases of IVDH (positive cases), but no signal in negative cases. ELISA values were significantly higher in the positive cases than in the negative cases. Both COMP protein and mRNA were present at high levels in the gray matter of the spinal cord in all species. CONCLUSION: In dog IVDH, release of COMP from the spinal cord in association with injury may lead to COMP accumulation in L-CSF posterior to the site of disc extrusion, and therefore might be a predictive marker of spinal cord injury.

Neuronal pentraxin receptor in cerebrospinal fluid as a potential biomarker for neurodegenerative diseases.

Neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by progressive loss of cognitive function, dementia, and problems with movements. In order to find new protein biomarkers of high specificity from cerebrospinal fluid (CSF) of AD and PD patients, one-dimensional gel electrophoresis (1-DE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as well as 2-DE analysis were performed. In 1-DE and LC-MS/MS 371 proteins were identified, among which levels of proteins such as isoform 1 of contactin-1, contactin-2, carnosine dipeptidase 1 (CNDP1), 120 kDa isoform precursor of neural cell adhesion molecule 1 (NCAM-120), alpha-dystroglycan, secreted protein acidic and rich in cysteine-like protein 1 precursor (SPARCL1), isoform 2 of calsyntenin 1 (CLSTN1), and neuronal pentraxin receptor (NPR) showed significant changes in AD or PD CSF compared with normal subjects. In 2-DE analysis approximately 747-915 spots were detected in CSF of AD or PD patients, from which 17-24 proteins with more than a 1.2 fold change were identified by tandem MS. Most proteins identified showed consistent changes in LC-MS/MS and 2-DE analysis. Three proteins that showed significant changes were selected for further validation by Western blot analysis. While NCAM-120 and alpha-dystroglycan exhibited higher levels in both AD and PD CSF compared with normal subjects, the level of NPR was increased only in AD CSF in Western blot analysis. The results were consistent with quantitative analysis of 2-DE spots. A higher level of NPR was also found in AD serum. This study suggests that NCAM-120, alpha-dystroglycan, and NPR are candidate biomarkers in CSF for neurodegenerative diseases, and that the changes in the CSF level of NPR may be specific for AD.

Reelin expression and glycosylation patterns are altered in Alzheimer's disease.

Reelin is a glycoprotein that is essential for the correct cytoarchitectonic organization of the developing CNS. Its function in the adult brain is less understood, although it has been proposed that Reelin is involved in signaling pathways linked to neurodegeneration. Here we analyzed Reelin expression in brains and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients and nondemented controls. We found a 40% increase in the Reelin protein levels in the cortex of AD patients compared with controls. Similar increases were detected at the Reelin mRNA transcriptional level. This expression correlates with parallel increases in CSF but not in plasma samples. Next, we examined whether CSF Reelin levels were also altered in neurological diseases, including frontotemporal dementia, progressive supranuclear palsy, and Parkinson's disease. The Reelin 180-kDa band increased in all of the neurodegenerative disorders analyzed. Moreover, the 180-kDa Reelin levels correlated positively with Tau protein in CSF. Finally, we studied the pattern of Reelin glycosylation by using several lectins and the anti-HNK-1 antibody. Glycosylation differed in plasma and CSF. Furthermore, the pattern of Reelin lectin binding differed between the CSF of controls and in AD. Our results show that Reelin is up-regulated in the brain and CSF in several neurodegenerative diseases and that CSF and plasma Reelin have distinct cellular origins, thereby supporting that Reelin is involved in the pathogenesis of a number of neurodegenerative diseases.

Genetic variability in the extracellular matrix protein as a determinant of risk for developing HTLV-I-associated neurological disease.

Aggrecan, which is a well-known proteoglycan in joint cartilage, also exists in the spinal cord and plays an important role in maintaining water content in the extracellular matrix structure. In this study, we first examined the variable number of tandem repeat (VNTR) polymorphism of the aggrecan gene in 227 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, in 217 HTLV-I-infected healthy carriers (HCs), and in 85 normal controls. The VNTR allele 28 (1,630 bp) was more frequently observed in HAM/TSP patients than in HCs (chi2=12.02, p=0.0005, odds ratio 1.79, 95% C.I. 1.29-2.50) and in controls (chi2=13.43, p=0.0002, odds ratio 2.54, 95% C.I. 1.52-4.25), although this allele was not related to disease progression or to HTLV-I provirus load. We also found that the aggrecan concentration in cerebrospinal fluid (CSF) from rapidly progressive HAM/TSP patients was significantly higher than in slowly progressive patients (corrected p=0.0145) but not in infected non-inflammatory neurological other disease controls (OND) (corrected p=0.078). We then analyzed this aggrecan VNTR polymorphism in the different set of patients with HAM/TSP (n=58) and healthy carriers (n=70). This analysis, again, revealed that allele 28 was detected more frequently in HAM/TSP group than in HCs (chi2=11.03, p=0.0009, odd ratio 3.04, 95% C.I. 1.55-5.97). The reproducibility of our study was regarded as a second- or third-class association by comparing combined p values and the Better Associations for Disease and GEnes (BADGE) system. Our results suggest that aggrecan polymorphism can be a novel genetic risk factor for developing HAM/TSP.

Altered levels of cerebrospinal fluid reelin in frontotemporal dementia and Alzheimer's disease.

Reelin is an essential glycoprotein for correct cytoarchitectonic organization during CNS development. Its function in the adult brain is far less well understood, but altered brain and blood reelin levels have been reported in some psychiatric disorders, and the possibility has been considered of an involvement of the reelin signaling pathway in neurodegeneration. Here we report, for the first time, the presence of detectable levels of reelin in rat and human cerebrospinal fluid (CSF) and show evidence for the involvement of a 180-kDa reelin fragment in two neurodegenerative disorders. This fragment was analyzed by Western blotting in CSF samples from 13 healthy control individuals and 14 frontotemporal dementia (FTD) and 20 Alzheimer's disease (AD) patients. Increased CSF 180-kDa reelin was found in FTD (161.7 +/- 6.7 arbitrary units; a.u.) and AD (151.4 +/- 3.8 a.u.) compared with control individuals (141.4 +/- 1.2 a.u., P < 0.05). Our results strongly suggest the involvement of reelin signaling in neurodegenerative pathologies.

Characterization of the various forms of the Reelin protein in the cerebrospinal fluid of normal subjects and in neurological diseases.

Reelin is a large extracellular glycoprotein that is defective in reeler mutant mice and plays a well-established role during brain development in human as well as rodents. In the adult brain, Reelin is expressed in a subset of GABAergic interneurons. Its role in disease states is not clearly defined, although it is implicated in autism and psychoses such as schizophrenia. In this report, we show that Reelin immunoreactive proteins can be detected in the human cerebrospinal fluid (CSF) with monoclonal antibodies directed against the N- and C-terminal regions of the protein. In CSF, Reelin is present as different products due to processing at two main sites; preservation at -20 degrees C increases processing further. CSF Reelin originates from the brain tissue and not from plasma. The protein was detected in comparable concentrations in children and adults, and the signal varied largely from subject to subject with no obvious correlation with age or neurological disease state.