Induction of cell death in rat brain by a gliotoxic factor from cerebrospinal fluid in multiple sclerosis.

The cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients contains a 17 kDa glycoproteic factor with gliotoxic properties in vitro. In order to study the physiopathological role of this gliotoxic factor in vivo, we have injected a partially purified preparation and appropriate controls in rat CSF and investigated whether it induces cell death in the rat central nervous system (CNS), 10 days and 3 months after injection. We used the TUNEL assay in association with specific immunohistochemistry to characterize dying cells in the gliotoxic factor- treated rat CNS. At 10 days post-injection, TUNEL-positive cells were observed in the whole rat CNS. They were particularly numerous in the choroid plexus, ependymal epithelium, cerebral white matter, cerebral vascular endothelium, arachnoid spaces and less frequent in the gray matter of brain and spinal cord. The predominant type of TUNEL-positive cells observed at 10 days post-injection was astrocytes, in white matter, gray matter, occasionnally around damaged endothelial cells in periventricular and subpial spaces. Other TUNEL-positive cells were identified as oligodendrocytes by an oligodendrocyte specific RIP immunostaining, at 10 days post-treatment with the gliotoxic factor. Interestingly, demyelination and death of oligodendrocytes were more important 3 months post-injection: TUNEL-RIP positive oligodendrocytes were generally associated with multifocal demyelinating areas. Clearly, the 17 kDa gliotoxic factor injection in rat CSF triggers demyelination and may be used as a new animal model for MS. Also, our results suggest a new possible scenario for MS pathogenesis: death of oligodendrocytes and astrocytes, stimulated by the MS gliotoxic factor causes the breakdown of the blood-brain barrier (BBB) and the demyelinating cascade.     

Complement regulator factor H in multiple sclerosis

A recent proteomic study published in this journal demonstrated lower cerebrospinal fluid (CSF) expression of factor H (fH), an important complement regulator, along with two other complement proteins, in active multiple sclerosis (MS) patients. We have previously demonstrated raised serum fH levels in MS and here, an extended analysis, quantifying fH in CSF, demonstrates no change in fH levels in active disease, but significantly raised levels in progressive disease. These findings support our previous work showing raised serum fH in patients with progressive MS, and our results predict that CSF fH levels will be raised rather than reduced in active disease.     

Tumor necrosis factor-alpha and its soluble receptors in plasma and cerebrospinal fluid of multiple sclerosis patients treated with methylprednisolone.

Demyelination is the main pathological feature of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. Tumor necrosis factor-alpha (TNF-alpha) can cause myelin damage and contribute to MS pathogenesis. We measured plasma and cerebrospinal fluid (CSF) levels of TNF-alpha and its soluble receptors, TNF-sRp55 and TNF-sRp75, in 18 patients with active MS, and in neurological and healthy controls. The same determinations were repeated on plasma and on CSF samples that were collected after the MS patients had ended a six-day treatment with high-dose methylprednisolone (MP). Pre- and post-treatment plasma and CSF TNF-alpha levels, when detectable, and those of TNF-sRp75, did not vary, and were similar to those of controls. CSF TNF-sRp55 levels were higher in acute MS patients than in controls. Post-treatment CSF TNF-sRp55 levels were higher than in the active phase of the disease. The MS patients, who clinically improved, tended to have the highest CSF TNF-sRp55 levels. The increase was due to intrathecal TNF-sRp55 synthesis. Although it is involved in MS pathogenesis, TNF-alpha is not detectable in plasma or in CSF samples from MS patients in various phases of the disease. A better marker of disease activity seems to be CSF TNF-sRp55 levels. The increased CSF levels of TNF-sRp55 in response to MP circumstantially suggest that this receptor could partially account for the beneficial effects of MP in acute MS.     

Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer's disease

We investigated the lipoprotein distribution and composition in cerebrospinal fluid (CSF) in a group of patients with Alzheimer's disease (AD) or affected by other types of dementia in comparison to non-demented controls. We found slightly decreased apolipoprotein (apo)E and cholesterol concentrations in CSF of AD patients and moderately increased apoA-I concentrations, while in patients suffering from other types of dementia the apoA-I CSF concentration was increased. ApoA-IV concentrations varied widely in human CSF, but were not associated with any clinical condition. HDL(2)-like apoE-containing lipoproteins represent the major lipoprotein fraction. In CSF of normal controls, only a minor HDL(3)-like apoA-I-containing lipoprotein fraction was observed; this fraction was more prevalent in AD patients. ApoA-II was recovered mostly in the HDL(3) density range, while apoA-IV was not associated with lipoproteins but appeared in a lipid-free form, co-localizing with LCAT immunoreactivity. Bi-dimensional analysis demonstrated pre-beta and alpha apoA-I-containing particles; apoE and apoA-II were detected only in alpha-migrating particles. ApoA-IV distributed both to pre-beta and gamma-migrating particles; the LCAT signal was co-localized in this gamma-migrating fraction. Enzymatically active LCAT was present in human CSF as well as PLTP activity and mass; no CETP mass was detected. In CSF from AD patients, LCAT activity was 50% lower than in CSF from normal controls. CSF lipoproteins induced a significant cholesterol efflux from cultured rat astrocytes, suggesting that they play an active role in maintaining the cholesterol homeostasis in brain cells.     

Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that high-affinity anti-DNA antibodies were one of the major components of the intrathecal IgG response in multiple sclerosis (MS) patients [Williamson et al., PNAS, 2001]. Recently we have shown that IgGs from the sera of MS patients are active in the hydrolysis of DNA. Here we have shown, for the first time, that average concentration of total proteins (132-fold), total IgGs (194-fold) and anti-DNA antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (CSF) of fifteen MS patients. The relative activities of total protein from sera and CSFs varied remarkably from patient to patient. It was surprising that the specific DNase activity of the total protein of CSF reparations were 198-fold higher than the serum ones. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous antibodies from CSF is unpredictably ∼49-fold higher than that from the sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that DNase IgGs of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and MS pathogenesis development.     

Characterisation of the expression of NMDA receptors in human astrocytes

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.     

Increased levels of tumor necrosis alpha and soluble vascular endothelial adhesion molecule-1 in the cerebrospinal fluid of patients with connective tissue diseases and multiple sclerosis

The aim of the present study was to investigate the serum and cerebrospinal fluid (CSF) concentrations of tumor necrosis factor alpha (TNF-alpha) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with primary progressive form of multiple sclerosis (MS) and in patients with connective tissue diseases (CTDs) complicated with central nervous system (CNS) involvement. Stimulation of sVCAM-1 release by TNF-alpha was demonstrated on endothelial cells of brain vessels. We intended to present the TNF-alpha stimulated elevation of sVCAM-1 in the serum and CSF in any cases of CNS lesion. Fifty patients with several CTDs complicated with neuropsychiatric symptoms and 25 MS patients with primary chronic progressive form of the disease were selected. Determinations of TNF-alpha and sVCAM-1 were performed using ELISA methods. TNF-alpha and sVCAM-1 concentrations were elevated in the CSF of all patients, intrathecal synthesis of sVCAM-1 was demonstrated in MS patients. The changes in the TNF-alpha and sVCAM-1 concentrations were independent from the clinical manifestations, immunoserological changes and quality of neuropsychiatric symptoms of the CTDs. The stimulatory effect of TNF-alpha was more pronounced in the CSF of MS patients.     

Comparison of Antibodies Hydrolyzing Myelin Basic Protein from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that antibodies (Abs) against myelin basic protein (MBP) are the major components of the antibody response in multiple sclerosis (MS) patients. We have recently shown that IgGs from sera of MS patients are active in the hydrolysis of MBP. However, in literature there are no available data concerning possible MBP-hydrolyzing Abs in cerebrospinal fluid (CSF) of MS patients. We have shown that the average content of IgGs in their sera is about 195-fold higher than that in their CSF. Here we have compared, for the first time, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4) in CSF and sera of MS patients. The average relative content of lambda-IgGs and kappa –IgGs in the case of CSFs (8.0 and 92.0%) and sera (12.3 and 87.7%) are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF - 40.4, 49.0, 8.2, and 2.5% of total IgGs, respectively and the sera - 53.6, 36.0, 5.6, and 4.8%, decreased in different order. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ∼54-fold higher than that of Abs from sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that anti-MBP abzymes of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development.     

Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.     

Comparative analysis of the CD19+ and CD138+ cell antibody repertoires in the cerebrospinal fluid of patients with multiple sclerosis

Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.     

Differential expression of complement proteins in cerebrospinal fluid from active multiple sclerosis patients

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with complex immunopathogenesis. Using the 2‐D DIGE technology, we separate CSF proteins from patients with active MS and control subjects. Three of the seven differential proteins identified were related with complement system, and the network analysis of the differential proteins revealed complement activation involvement in active MS. Complement C4b (gamma chain) was confirmed elevated by performing western blotting analysis (P < 0.01). The present results are an independent quantitative proteomic measure in CSF from active MS patients. The differential expression of the complement C4b and related proteins in CSF provides potential biomarkers as well as evidence for the involvement of complement activation in the pathogenesis of MS disease. J. Cell. Biochem. 112: 1930–1937, 2011. © 2011 Wiley‐Liss, Inc.     

An immunocytochemical study of normal and abnormal human cerebrospinal fluid with monoclonal antibodies to glial fibrillary acidic protein

The presence of astrocytes in the cerebrospinal fluid (CSF) of patients may be of diagnostic importance. However, the frequency with which astrocytes are shed into normal and abnormal human CSF is unknown. This issue was studied using monoclonal antibodies to an astrocyte-specific antigen, glial fibrillary acidic protein (GFAP), and immunoperoxidase cytochemistry. The study was prospectively conducted on 108 CSF preparations diagnosed as normal, reactive, metastatic malignancy or suspicious for metastatic malignancy. To validate these methods, cells from a clonal human glioma cell line, which contains astrocytes rich in GFAP, were processed in a manner identical to that used for the CSFs obtained from patients. Studies of the human glioma cell line demonstrated intense GFAP immunoreactivity in the majority of the malignant astrocytes. In contrast, none of the CSFs contained GFAP-positive cells. We conclude that immunocytochemical methods can detect GFAP in neoplastic human astrocytes but that nonneoplastic GFAP-positive cells are uncommon in human CSF; such cells were not seen in our large series of normal and abnormal human CSFs. The immunocytochemical detection of GFAP may be a useful criterion for distinguishing malignant astrocytes from other types of malignant cells in human CSF.     

Simultaneous Measurement of 2':3' Cyclic-Nucleotide 3' Phosphodiesterase and RNase Activities in Sera and Spinal Fluids of Multiple Sclerosis Patients

Abstract: The enzymes 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and RNase were simultaneously measured in the sera and CSF of multiple sclerosis (MS) and non-MS patients. No evidence of increased activity for these enzymes could be found regardless of pathology in either fluid source. Discrepancies between the present results and those from two previous studies that reported significant increases in CNPase activity in the CSF of patients with MS were carefully analyzed. It was concluded that the apparent increased CNPase activity correlated with MS in both previous studies was most probably the result of methodological and computational difficulties.     

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.     

Purification of a human urinary colony-stimulating factor

Colony-stimulating factor (CSF), a protein required for the in vitro formation of colonies composed of granulocytes and/or macrophages, was isolated from the urine of anemic patients by using a seven-step procedure. The purified, homogeneous CSF had a specific activity of 1.9 X 10(8) U/absorbance unit at 280 nm (AU). This represents an overall purification of 25,330-fold and a total recovery of 3.8%. Upon iodination of the protein, the radioactivity migrated on sodium dodecyl sulfate (SDS) gel electrophoresis as a single peak with an apparent molecular weight of 46,000; reduction with mercaptoethanol caused dissociation to a single component of molecular weight 23,000. Only the dimer is active in stimulating colony formation. Urinary CSF stimulates formation of colonies comprising only macrophages in the mouse bone marrow cell culture assay. A neutralizing antibody raised against mouse L-cell CSF did not neutralize the activity of the urinary CSF but did bind it. This may indicate that the relative positions of antibody binding sites and the active sites are different in these two glycoproteins.     

Evidence for Two Cholesterol Ester Hydrolases in Human Cerebrospinal Fluid

Abstract: In the present study, the properties, such as pH optima, detergent requirement, and effects of various lipids, of cholesterol ester hydrolase in human cerebrospinal fluid (CSF) were examined, and the activity levels of the enzyme in CSF from multiple sclerosis (MS) patients and non-MS individuals were compared. Our data indicate that the enzyme in CSF exhibits two pH optima: pH 6.0 in the presence of Triton X-100 and pH 7.0 in the presence of sodium taurocholate. Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) enhanced the hydrolase activity at pH 6.0. The activity at pH 7.0, on the other hand, was enhanced slightly in the presence of PE but was inhibited in the presence of PS. These data suggest the presence of two cholesterol ester hydrolases in CSF and also indicate that the activity at pH 6.0 may be due to microsomal enzyme in brain and that at pH 7.0 may be due to myelin enzyme. The hydrolase activity at pH 7.0 was significantly lower in CSF from MS patients. The activity at pH 6.0 in CSF from MS and non-MS patients, however, did not differ significantly. This indicates that the reduction in pH 7.0 hydrolase activity in CSF may be related to demyelination.     

Soluble interleukin-2 receptor and soluble CD8 molecules in cerebrospinal fluid and serum of patients with multiple sclerosis.

The purpose of this study was to analyse soluble interleukin-2 receptor (sIL-2R) and soluble CD8 (sCD8) molecules in the cerebrospinal fluid (CSF) and serum of 18 patients with definite multiple sclerosis (MS) and of 16 with noninflammatory neurologic diseases (NIND). All MS patients suffered from an exacerbation of the relapsing-remitting form of the disease within one month before examination. The mean serum levels of sIL-2R and sCD8 in the MS patients were not significantly different from those of NIND patients. Only one patient with MS had detectable sIL-2R in the CSF. CSF sCD8 was detectable in 10 of 18 MS patients and in 1 of 16 NIND patients. Our data indicate that the CSF and serum sIL-2R concentrations do not correlate with the disease activity. Conversely, increased levels of sCD8 only in the CSF of MS patients support the hypothesis of an intrathecal activation of CD8+ cells in MS. We think that CSF sCD8 can be a useful marker for the presence of activated T cells in the central nervous system.     

Gliotoxic action of glutamate on cultured astrocytes

Because of the well-documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L-glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L-glutamate resulted in a concentration- and time-dependent increase in the release of LDH. L-Glutamate-induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L-glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L-glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L-glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.