Cerebrospinal Fluid Protein Patterns Research Articles

Nusinersen Modulates Proteomics Profiles of Cerebrospinal Fluid in Spinal Muscular Atrophy Type 1 Patients.

Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular disorder caused by a survival motor neuron 1 gene (SMN1) mutation and characterized by progressive muscle weakness. Without supportive care, SMA type 1 is rapidly fatal. The antisense oligonucleotide nusinersen has recently improved the natural course of this disease. Here, we investigated, with a functional proteomic approach, cerebrospinal fluid (CSF) protein profiles from SMA type 1 patients who underwent nusinersen administration to clarify the biochemical response to the treatment and to monitor disease progression based on therapy. Six months after starting treatment (12 mg/5 mL × four doses of loading regimen administered at days 0, 14, 28, and 63), we observed a generalized reversion trend of the CSF protein pattern from our patient cohort to that of control donors. Notably, a marked up-regulation of apolipoprotein A1 and apolipoprotein E and a consistent variation in transthyretin proteoform occurrence were detected. Since these multifunctional proteins are critically active in biomolecular processes aberrant in SMA, i.e., synaptogenesis and neurite growth, neuronal survival and plasticity, inflammation, and oxidative stress control, their nusinersen induced modulation may support SMN improved-expression effects. Hence, these lipoproteins and transthyretin could represent valuable biomarkers to assess patient responsiveness and disease progression.

Codon 129 Polymorphism Specific Cerebrospinal Fluid Proteome Pattern in Sporadic Creutzfeldt−Jakob Disease and the Implication of Glycolytic Enzymes in Prion-Induced Pathology

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which can reflect a physiological and pathological state of the central nervous system. In our present study, we show CSF protein patterns from patients with the two most frequent subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) defined by the codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2). The densitometric analysis of 2D gels showed up-regulation of 27 and down-regulation of 3 proteins in the MM-sCJD as well as the up-regulation of 24 proteins in the VV-sCJD as compared to nondemented control. Almost 40% of sCJD specific regulated proteins in CSF are involved in glucose metabolism, regardless of the codon 129 polymorphism. The increase in CSF levels of lactate dehydrogenase (LDH), glucose-6-phosphate isomerase (G6PI), and fructose-bisphosphate aldolase A (ALDOA) were validated on a larger group of sCJD patients including three possible codon 129 polymorphism carriers and three control groups consisting of nondemented, neurological cases as well as patients suffering from Alzheimer's disease or vascular dementia. Subsequently, the abundance of these glycolytic enzymes in the brain as well as their cellular localization were determined. This study demonstrates for the first time the implication of G6PI in prion-induced pathology as well as its cellular translocalization in sCJD. The identification of sCJD-regulated proteins in CSF of living symptomatic patients in our study can broaden our knowledge about pathological processes occurring in sCJD, as they are still not fully understood.

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease-associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high-resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2-D protein gels. Using state-of-the-art 2-DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI-TOF/TOF MS for protein identification, a highly detailed 2-DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2-DE CSF maps before.

Cerebrospinal fluid protein findings in various lower back pain syndromes

Cerebrospinal fluid (CSF) total protein, albumin and IgG concentrations were measured in 53 patients with lower back pain syndromes. In the majority of the patients (81 %) the protein values were within normal ranges, a finding contrary to previous studies. In six of 28 patients with acute lumbar disc prolapse and in two of four cases with spinal stenosis, a clearly abnormal protein pattern was observed, while patients who had been earlier operated on for disc prolapse showed normal values. The calculated permeability indexes suggest that the elevation of various proteins is linked with increased permeability across the blood-brain barrier and that the local immunoglobulin synthesis in CNS is lacking. In only one of the cases with an abnormal CSF protein pattern could no evidence of the cause of the pathological finding be observed in subsequent examinations.

Use of albumin quotient and IgG index to differentiate blood‐ vs brain‐derived proteins in the cerebrospinal fluid of cats with feline infectious peritonitis

Background: Inflammation of the central nervous system (CNS) is a frequent condition in cats but etiology often remains unsolved. Routine cerebrospinal fluid (CSF) analysis can be extended through the calculation of the albumin quotient (Q alb), a marker of the integrity of the blood–brain barrier (BBB), and IgG index, an estimate of intrathecal IgG synthesis. Objectives: The purpose of this study was to validate nephelometric methods for CSF protein analysis, and to use the Q alb and IgG index to discriminate blood‐ and brain‐derived immunoglobulin fractions in cats with feline infectious peritonitis (FIP). Methods: Cats presented to our clinic between 2001 and 2005 were included in the study based on clinical and laboratory data and histopathologic findings at necropsy. Cats were grouped as having nonneurologic disease (controls; n=37), brain tumors (n=8), FIP involving the CNS (n=12), and extraneural FIP (n=12). CSF‐total protein (TP) was measured and albumin and IgG concentrations were measured in paired CSF/serum samples; Q alb and IgG index were calculated. Intraassay and interassay precision of the nephelometric assays were determined using pooled samples. Results: Coefficients of variation for the nephelometric assays ranged from 2.7% to 7.2%. In control cats, CSF‐TP concentration ranged from 0.06 to 0.36 g/L, Q alb ranged from 0.6 to 5.7 × 10−3, and IgG index ranged from 0.3 to 0.6. Q alb and IgG index were significantly higher in cats with brain tumors and cats with CNS‐FIP compared with other groups. Compared with control cats, pleocytosis was evident in 8 of 12 (67%) cats and CSF‐TP was increased in 3 of 12 (25%) cats with CNS‐FIP. Conclusion: Nephelometry is a reliable method for measurement of CSF protein, albumin, and IgG in cats. The Q alb and IgG index did not identify a CSF protein pattern specific for BBB dysfunction or intrathecal IgG synthesis in cats with CNS‐FIP.

Identifying Pharmacodynamic Protein Markers of Centrally Active Drugs in Humans: A Pilot Study in a Novel Clinical Model

Recognizing specific protein changes in response to drug administration in humans has the potential for significant utility in clinical research. In spite of this, many methodological and practical questions related to assessing such changes are unanswered. We conducted a series of clinical studies to assess the feasibility of measuring changes in proteins related to drug administration using a mass-spectrometry proteomics technique capable of quantifying hundreds of proteins simultaneously in cerebrospinal fluid (CSF) and plasma. Initially, the normal variability of proteins in these compartments was characterized in 16 healthy volunteers over a 2-week period. Drug-associated changes were subsequently assessed in the plasma and CSF proteomes of 11 subjects given atomoxetine, which served as a selective, centrally active probe to test the model. Protein levels in the CSF and plasma were unchanged between visits in the normal variability study. In contrast, statistically significant changes were detected in the CSF protein pattern after drug treatment. These studies suggest that identification of changes in the CSF proteome associated with the administration of centrally active drugs is feasible, and may be of value in the development of new drugs, as well as broader clinical research.

Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography‐Fourier transform ion cyclotron resonance mass spectrometry

This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.

Combined proteomic approach with SELDI-TOF-MS and peptide mass fingerprinting identified the rapid increase of monomeric transthyretin in rat cerebrospinal fluid after transient focal cerebral ischemia

Several proteins are known to be markedly expressed in the brain during cerebral ischemia, however the change in protein profiles within the cerebrospinal fluid (CSF) after an ischemic insult has not been fully elucidated. We studied the changes in the CSF proteome in rat transient middle cerebral artery occlusion model. Surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) was used to detect the time-course changes in CSF protein patterns after transient focal brain ischemia. According to hierarchical cluster analysis by self-organising tree algorism (SOTA), the temporal pattern of protein peaks was divided into four groups: acute increase group, chronic increase group, gradual decrease group and unchanged group. In the acute increase group, the expression of a 13.6-kDa protein was markedly increased during the acute phase. The 13.6-kDa protein was identified as monomeric form of transthyretin using two-dimensional electrophoresis and peptide mass fingerprinting based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The monomeric transthyretin may represent an ischemia-specific CSF marker to indicate the sequential changes according to ischemic insults of the brain.

Mercury and Proteins in Cerebrospinal Fluid in Subjects Exposed to Mercury Vapor

Mercury (Hg) and protein levels in cerebrospinal fluid (CSF), and mercury in plasma (P), erythrocytes (Ery), and urine (U), were determined in 10 workers exposed to mercury vapor for 2-28 (median 13) years and in 16 occupationally unexposed referents. CSF-Hg was analyzed using radiochemical neutron activation analysis and P-Hg, Ery-Hg, and U-Hg were analyzed using cold-vapor atomic absorption spectrometry. P-HB and U-Hg were significantly higher, but Ery-Hg was similar in the exposed workers (37 nmole/liter, 16 nmole/mmole creatinine, and 56 nmole/liter, respectively) compared with the referents (7.1 nmole/liter, 1.9 nmole/mmole creatinine, and 52 nmole/liter, respectively). CSF-Hg was correlated to P-Hg, and in workers with current high exposure (P-Hg > 50 nmole/liter), the CSF-Hg was significantly higher than in the reference group (1.08 versus 0.35 nmole/liter; P = 0.002). In two individuals, studied after ceased occupational exposure, a decrease of CSF-Hg was seen. There were no indications of changes in the CSF protein pattern in the exposed workers.

Late onset globoid leukodystrophy: unusual clinical and CSF findings

During a febrile upper respiratory tract illness this 4 year old boy developed left hemiparesis, which progressed to loss of walking and even of sitting finally to tetraplegia. The cerebrospinal fluid protein pattern showed blood-brain barrier damage with additional intrathecal IgG synthesis. The symptoms responded to steroid therapy but resumed and worsened on withdrawal. Only late, when visual evoked potentials and nerve conduction velocity proved to be impaired, was Krabbe disease diagnosed on the assay of cultured fibroblasts for galactocerebroside-beta-galactosidase. We discuss the significance of possible endogenous production of IgG in the CNS.

Cerebrospinal Fluid Protein Profile in Experimental Pneumococcal Meningitis and Its Alteration by Ampicillin and Anti-Inflammatory Agents

We studied the effects of ampicillin and anti-inflammatory agents on cerebrospinal fluid (CSF) protein patterns in rabbits with meningitis caused by Streptococcus pneumoniae. CSF proteins were analyzed in the acute phase of infection and during convalescence by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by two types of staining or by immunoblotting. During the acute phase a massive influx of serum albumin into the CSF was accompanied by the appearance of other proteins of high and low molecular weight. In untreated animals and animals given ampicillin alone or together with indomethacin, the abnormal pattern persisted for up to 30 d. Combined treatment with ampicillin and dexamethasone or oxindanac, a new nonsteroidal anti-inflammatory drug, partially prevented the alterations in CSF protein patterns. Reversion to normal CSF protein patterns was fastest in these two groups.

The intrathecal immune response in the early stage of multiple sclerosis

Sequential pairs of cerebrospinal fluid (CSF) and serum samples from 10 patients followed for 2.5–12 years after onset of unilateral optic neuritis (ON) were studied. Eight patients developed definite multiple sclerosis (MS) during the observation period. All patients had normal CSF protein patterns on agar or agarose gel electrophoresis at onset. Six patients developed oligoclonal immunoglobulin (Ig) bands in the CSF during the observation period. Imprint immunofixation of electrofocused specimens disclosed intrathecal synthesis of oligoclonal IgG antibodies to 1 or more of 6 viruses (measles, herpes simplex type 1, varicella-zoster, cytomegalo, mumps, rota) during the observation period in 8 patients. Changes in patterns of intrathecally synthesized viral antibodies, characterized by the appearance of “new” antibody populations and the waxing or waning of others were observed in 6 patients. The results suggest that the early stage of MS in some patients is associated with transient as well as permanent recruitment of B cell clones producing viral antibodies of different specificities.

Electrophoresis and immunoblot of cerebrospinal fluid proteins in spasmodic torticollis.

Protein patterns of cerebrospinal fluid (CSF) from patients with spasmodic torticollis (ST) were investigated to determine whether abnormalities previously reported could be detected and further identified. CSF was collected from 12 patients with ST and 6 normal controls. The CSF proteins were analyzed using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and silver staining. In 11 of the 12 patients with ST, a CSF protein pattern was observed which differed from that in the controls. The identity of the abnormal proteins was ascertained by blotting and immunostaining with specific antisera to IgG and ceruloplasmin (Cp). CSF from 2 of 12 patients had distinct bands staining for IgG and 7 had abnormal immunostaining for Cp.

Effect of sample preparation on cerebrospinal fluid protein patterns in polyacrylamide gels.

The method of preparing CSF and dilute serum samples for polyacrylamide gel electrophoresis has a marked effect on the pattern of the resolved proteins. The incorporation of sucrose or glycerol into the sample seriously impairs the quality of the resolution and affects the actual number of bands resolved and the relative mobility of certain proteins. The use of sucrose as an anti-convection medium in dense samples such as serum and CSF is not recommended.

Quantitation of plasma proteins in cerebrospinal fluid.

Summary. Fifteen plasma proteins were determined both in unconcentrated cerebrospinal fluid (CSF) and in serum from 22 patients suffering from various psychiatric and neurological disorders, by means of rocket immunoelectrophoresis. The procedure is described and the values are reported. The concentration of the individual proteins in the CSF is correlated to the concentration in serum and to the total protein content of the CSF. It is concluded that the CSF protein pattern can only be evaluated if the CSF total protein is known and if the plasma protein pattern in blood is investigated by means of, e.g., an electrophoresis in agarose gel.