Increased CSF Levels of Apolipoproteins and Complement Factors in Trigeminal Neuralgia Patients-In Depth Proteomic Analysis Using Mass Spectrometry.

The main cause of trigeminal neuralgia (TN) is compression of a blood vessel at the root entry zone of the trigeminal nerve. However, a neurovascular conflict does not seem to be the only etiology and other mechanisms are implicated in the development of the disease. We hypothesized that TN patients may have distinct protein expression in the CSF. In this study, lumbar CSF from TN patients (n = 17), scheduled to undergo microvascular decompression, and from controls (n = 20) was analyzed and compared with in depth mass spectrometry TMTbased quantitative proteomics. We identified 2552 unique proteins, of which 46 were significantly altered (26 increased, and 20 decreased, q-value < .05) in TN patients compared with controls. An over-representation analysis showed proteins involved in high-density lipoprotein, such as Apolipoprotein A4, Apolipoprotein M, and Apolipoprotein A1, and the extracellular region, including proteins involved in the complement cascade to be over-represented. We conclude that TN patients have distinct protein expression in the CSF compared to controls. The pathophysiological background of the protein alterations found in this study warrants further investigation in future studies. PERSPECTIVE: In this article, cerebrospinal fluid from patients with trigeminal neuralgia was analyzed using in depth shotgun proteomics, revealing 46 differentially expressed proteins compared to controls. Among these, apolipoproteins and proteins involved in the complement system were elevated and significantly over-represented, implying an inflammatory component in the pathophysiology of the disease.

Subtypes Based on Six Apolipoproteins in Non-Demented Elderly Are Associated with Cognitive Decline and Subsequent Tau Accumulation in Cerebrospinal Fluid.

Apolipoproteins (APOs) have been implicated in the pathogenesis of Alzheimer's disease (AD). In the present study, we aimed to investigate if patterns of cerebrospinal fluid (CSF) APOs (APOA-I, APOC-III, APOD, APOE, APOH, and APOJ) levels are associated with changes over time in cognition, memory performance, neuroimaging markers, and AD-related pathologies (CSF Aß42, t-tau, and p-tau) in non-demented older adults. At baseline, a total of 241 non-demented older adults with CSF APOs data was included in the present analysis. Hierarchical agglomerative cluster analysis including the six CSF APOs was carried out. Among non-demented older adults, we identified two clusters. Compare with the first cluster, the second cluster had higher levels of APOs in CSF. Additionally, the second cluster showed a more benign disease course, including slower cognitive decline and slower p-tau accumulation in CSF. Our data highlight the importance of APOs in the pathogenesis of AD.

α-synuclein-lipoprotein interactions and elevated ApoE level in cerebrospinal fluid from Parkinson's disease patients.

The progressive accumulation, aggregation, and spread of α-synuclein (αSN) are common hallmarks of Parkinson's disease (PD) pathology. Moreover, numerous proteins interact with αSN species, influencing its toxicity in the brain. In the present study, we extended analyses of αSN-interacting proteins to cerebrospinal fluid (CSF). Using coimmunoprecipitation, followed by mass spectrometry, we found that αSN colocalize with apolipoproteins on lipoprotein vesicles. We confirmed these interactions using several methods, including the enrichment of lipoproteins with a recombinant αSN, and the subsequent uptake of prepared vesicles by human dopaminergic neuronal-like cells. Further, we report an increased level of ApoE in CSF from early PD patients compared with matched controls in 3 independent cohorts. Moreover, in contrast to controls, we observed the presence of ApoE-positive neuromelanin-containing dopaminergic neurons in substantia nigra of PD patients. In conclusion, the cooccurrence of αSN on lipoprotein vesicles, and their uptake by dopaminergic neurons along with an increase of ApoE in early PD, proposes a mechanism(s) for αSN spreading in the extracellular milieu of PD.

Cholesterol and markers of cholesterol turnover in multiple sclerosis: relationship with disease outcomes.

Multiple sclerosis (MS) is a chronic central nervous system disease that is associated with progressive loss of myelin and subsequent axonal degeneration. Cholesterol is an essential component of mammalian cellular and myelin membranes. In this systematic review, we examined the relationship between levels of cholesterol and markers of cholesterol turnover in circulation and/or cerebrospinal fluid (CSF) and disease outcomes in adults with clinically isolated syndrome (CIS) or confirmed MS. Studies suggest that elevated levels of circulating low density lipoprotein cholesterol (LDL), total cholesterol, and particularly, apolipoprotein B and oxidized LDL are associated with adverse clinical and MRI outcomes in MS. These relationships were observed as early as CIS. The studies also suggest that oxysterols, cholesterol precursors, and apolipoprotein E may be markers of specific disease processes in MS, but more research is required to elucidate these processes and relationships. Taken together, the data indicate that cholesterol and markers of cholesterol turnover have potential to be used clinically as biomarkers of disease activity and may even be implicated in the pathogenesis of MS.

Protective associations of HDL with blood-brain barrier injury in multiple sclerosis patients.

The purpose of this work was to investigate the associations of serum cholesterol and apolipoproteins with measures of blood-brain barrier (BBB) permeability and CNS inflammation following the first clinical demyelinating event. This study included 154 patients [67% female; age, 29.5 ± 8.2 years (mean ± SD)] enrolled in a multi-center study of interferon ß1-a treatment following the first demyelinating event. Blood and cerebrospinal fluid (CSF) were obtained at screening prior to treatment. A comprehensive serum lipid profile and multiple surrogate markers of BBB breakdown and CNS immune activity were obtained. Higher levels of serum HDL cholesterol (HDL-C) and ApoA-I were associated with lower CSF total protein level, CSF albumin level, albumin quotient, and CSF IgG level (all P ≤ 0.001 for HDL-C and all P < 0.01 for ApoA-I). HDL-C was also associated with CSF CD80+ (P < 0.001) and with CSF CD80+CD19+ (P = 0.007) cell frequencies. Higher serum HDL is associated with lower levels of BBB injury and decreased CD80+ and CD80+CD19+ cell extravasation into the CSF. HDL may potentially inhibit the initiation and/or maintenance of pathogenic BBB injury following the first demyelinating event.

Effects of cerebrospinal fluid proteins on brain atrophy rates in cognitively healthy older adults.

Biomarkers associated with Alzheimer's disease (AD)-like brain atrophy in healthy individuals may identify mechanisms involved in early stage AD. Aside from cerebrospinal fluid (CSF) ß-amyloid42 (Aß42) and tau, no studies have tested associations between CSF proteins and AD-like brain atrophy. We studied 90 healthy elders, who underwent lumbar puncture at baseline, and serial magnetic resonance imaging scans for up to 4 years. We tested statistical effects of baseline CSF proteins (N = 70 proteins related to Aß42-metabolism, microglial activity, and synaptic/neuronal function) on atrophy rates in 7 AD-related regions. Besides the effects of Aß42 and phosphorylated tau (P-tau) that were seen in several regions, novel CSF proteins were found to have effects in inferior and middle temporal cortex (including apolipoprotein CIII, apolipoprotein D, and apolipoprotein H). Several proteins (including S100ß and matrix metalloproteinase-3) had effects that depended on the presence of brain Aß pathology, as measured by CSF Aß42. Other proteins (including P-tau and apolipoprotein D) had effects even after adjusting for CSF Aß42. The statistical effects in this exploratory study were mild and not significant after correction for multiple comparisons, but some of the identified proteins may be associated with brain atrophy in healthy persons. Proteins interacting with CSF Aß42 may be related to Aß brain pathology, whereas proteins associated with atrophy even after adjusting for CSF Aß42 may be related to Aß-independent mechanisms.

Translational spatial task and its relationship to HIV-associated neurocognitive disorders and apolipoprotein E in HIV-seropositive women.

HIV-associated neurocognitive disorders (HAND) continue to be a neurological complication of HIV infection in the era of combined antiretroviral therapy. Hippocampal neurodegeneration and dysfunction occurs as a result of HIV infection, but few studies to date have assesses spatial learning and memory function in patients with HAND. We used the Memory Island (MI) test to study the effects of HIV infection, apolipoprotein E (ApoE) allele status, and cerebral spinal fluid (CSF) ApoE protein levels on spatial learning and memory in our cohort of Hispanic women. The MI test is a virtual reality-based computer program that tests spatial learning and memory and was designed to resemble the Morris Water Maze test of hippocampal function widely used in rodent studies. In the current study, HIV-seropositive women (n = 20) and controls (n = 16) were evaluated with neuropsychological (NP) tests, the MI test, ApoE, and CSF ApoE assays. On the MI, the HIV-seropositive group showed significant reduced learning and delayed memory performance compared with HIV-seronegative controls. When stratified by cognitive performance on NP tests, the HIV-seropositive, cognitively impaired group performed worse than HIV-seronegative controls in ability to learn and in the delayed memory trial. Interestingly, differences were observed in the results obtained by the NP tests and the MI test for ε4 carriers and noncarriers: NP tests showed effects of the ε4 allele in HIV-seronegative women but not HIV-seropositive ones, whereas the converse was true for the MI test. Our findings suggest that the MI test is sensitive in detecting spatial deficits in HIV-seropositive women and that these deficits may arise relatively early in the course of HAND.

Upcoming candidate cerebrospinal fluid biomarkers of Alzheimer's disease.

Dementia due to Alzheimer's disease (AD) is estimated to reach epidemic proportions by the year 2030. Given the limited accuracy of current AD clinical diagnosis, biomarkers of AD pathologies are currently being sought. Reductions in cerebrospinal fluid levels of ß-amyloid 42 (a marker of amyloid plaques) and elevations in tau species (markers of neurofibrillary tangles and/or neurodegeneration) are well-established as biomarkers useful for AD diagnosis and prognosis. However, novel markers for other features of AD pathophysiology (e.g., ß-amyloid processing, neuroinflammation and neuronal stress/dysfunction) and for other non-AD dementias are required to improve the accuracy of AD disease diagnosis, prognosis, staging and therapeutic monitoring (theragnosis). This article discusses the potential of several promising novel cerebrospinal fluid analytes, highlights the next steps critical for advancement in the field, and provides a prediction on how the field may evolve in 5-10 years.

Can apolipoproteins and complement factors be biomarkers of Alzheimer's disease?

Alzheimer's disease is the most common cause of dementia in elderly persons. Quick diagnosis of Alzheimer's disease will allow treatments that may help slow its progression. The correlation between cerebrospinal fluid (CSF) parameters and progression of Alzheimer's disease is higher than and independent of other risk factors. We have compared sixteen CSF samples of clinically diagnosed Alzheimer's disease patients with non demented subjects using proteomics approach. Apolipoprotein E, apolipoprotein J, complement C4b, hemopexin and complement factor B were identified as differentially expressed proteins. Pathway analyses show that these proteins have interacting partners in Alzheimer's and apoptotic pathways. The possible roles of these proteins in relation to the disease are discussed.

CSF multianalyte profile distinguishes Alzheimer and Parkinson diseases.

The therapeutic imperative for Alzheimer disease (AD) and Parkinson disease (PD) calls for discovery and validation of biomarkers. Increased cerebrospinal fluid (CSF) tau and decreased amyloid (A) beta42 have been validated as biomarkers of AD. In contrast, there is no validated CSF biomarker for PD. We validated our proteomics-discovered multianalyte profile (MAP) in CSF from 95 control subjects, 48 patients with probable AD, and 40 patients with probable PD. An optimal 8-member MAP agreed with expert diagnosis for 90 control subjects (95%), 36 patients with probable AD (75%), and 38 patients with probable PD (95%). This MAP consisted of the following (in decreasing order of contribution): tau, brain-derived neurotrophic factor, interleukin 8, Abeta42, beta2-microglobulin, vitamin D binding protein, apolipoprotein (apo) AII, and apoE. This first large-scale validation of a proteomic-discovered MAP suggests a panel of 8 CSF proteins that are highly effective at identifying PD and moderately effective at identifying AD.

Analysis of potential diagnostic biomarkers in cerebrospinal fluid of idiopathic normal pressure hydrocephalus by proteomics.

BACKGROUND: The pathogenesis of idiopathic normal pressure hydrocephalus (INPH) is unknown, and the syndrome of INPH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis and treatment of INPH and thus to study their role in the disease process. METHODS: A comparative proteomic analysis was used for clinical screening of cerebrospinal fluid (CSF) proteins in 15 patients with INPH and compared with 12 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with CSF proteins between individual INPH patients and controls. RESULTS: Seven proteins and their isoforms, including leucine-rich alpha-2-glycoprotein (LRG), alpha1-antichymotrypsin, apolipoprotein D, apolipoprotein J, haptoglobin alpha1, serum albumin, and alpha-1-microglobulin/bikunin precursor showed significant changes in CSF of INPH patients compared with controls by proteomic analysis. And significant higher CSF levels of LRG in INPH patients compared with controls were found by ELISA. CONCLUSIONS: These results indicate that there are significant differences in the expression of certain proteins in the CSF of patients with INPH and normal subjects. In particular, the CSF level assay of LRG suggests that LRG is a specific biomarker for INPH and has potential use in the diagnosis and indication for CSF shunting.

Mammalian embryonic cerebrospinal fluid proteome has greater apolipoprotein and enzyme pattern complexity than the avian proteome.

During early stages of embryo development, the brain cavity is filled with Embryonic Cerebro-Spinal Fluid, which has an essential role in the survival, proliferation and neurogenesis of the neuroectodermal stem cells. We identified and analyzed the proteome of Embryonic Cerebro-Spinal Fluid from rat embryos (Rattus norvegicus), which includes proteins involved in the regulation of Central Nervous System development. The comparison between mammalian and avian Embryonic Cerebro-Spinal Fluid proteomes reveals great similarity, but also greater complexity in some protein groups. The pattern of apolipoproteins and enzymes in CSF is more complex in the mammals than in birds. This difference may underlie the greater neural complexity and synaptic plasticity found in mammals. Fourteen Embryonic Cerebro-Spinal Fluid gene products were previously identified in adult human Cerebro-Spinal Fluid proteome, and interestingly they are altered in patients with neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis may contribute to our understanding of Central Nervous System development and evolution, and these human diseases.

Proteomic studies of potential cerebrospinal fluid protein markers for Alzheimer's disease.

By comparing the cerebrospinal fluid (CSF) proteome between Alzheimer's disease (AD) patients and controls, it may be possible to identify proteins that play a role in the disease process and thus to study the pathogenesis of AD. Two-dimensional gel electrophoresis (2-DE), SYPRO Ruby staining and mass spectrometry were used for clinical screening of disease-influenced CSF proteins in AD patients compared to controls. In order to increase the detection of CSF proteins and to improve the separation of protein isoforms in a 2-D gel, micro-narrow range IPG strips were used. The levels of eight proteins and their isoforms, including apolipoprotein A1, apolipoprotein E, apolipoprotein J, beta-trace, retinol-binding protein, kininogen, alpha-1 antitrypsin, cell cycle progression 8 protein, and alpha-1beta glycoprotein were significantly altered in CSF of AD patients compared to controls. Furthermore, we also used liquid-phase IEF, as a prefractionation step, prior to 2-DE for comparison of CSF proteins between individual AD patients and controls. The levels of 37 proteins spots were altered in AD patients. One of the identified proteins, alpha-2-HS glycoprotein, has not previously been linked to AD. Our study shows that several glycoproteins are altered in AD.

The levels of soluble amyloid beta in different high density lipoprotein subfractions distinguish Alzheimer's and normal aging cerebrospinal fluid: implication for brain cholesterol pathology?

Several previous studies reported the association of the soluble form of amyloid beta (sA beta) protein, a major constituent of amyloid deposits in Alzheimer's disease (AD), with normal blood, cerebrospinal fluid (CSF) and central nervous system high density lipoproteins (HDLs). The present report aimed to elucidate the pattern of sA beta and apolipoprotein (apo) distribution in AD CSF-HDL subfractions. We studied AD CSF-HDL subfractions by SDS/PAGE and immunoblot analysis after CSF fractionation via density flotation ultracentrifugation. AD CSF was characterized by (i) increased sA beta and apo content of the HDL(1), and (ii) sA beta association with apoE and apoJ in HDL(2), HDL(3) and very high density lipoproteins. The finding supports our proposed hypothesis that upregulation of brain cholesterol dynamics is a fundamental event in the pathophysiology of AD and that sA beta binding to apo and lipid may have important structure-functional consequences.

Increased intrathecal production of apolipoprotein D in multiple sclerosis.

Apolipoprotein D (apoD) is a small glycoprotein responsible for the local transport of small hydrophobic ligands. Within the nervous system, apoD may be an acute phase protein that is upregulated in a variety of neuropathological conditions and is involved in the removal of lipids during nerve cell degeneration and provision of lipids during the regenerative phase. In this study, we measured cerebrospinal fluid (CSF) and serum apoD levels in patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré Syndrome (GBS), infectious inflammatory neurological diseases (IND) and non-inflammatory neurological diseases (NND). We found that mean CSF apoD levels are significantly increased in patients with CIDP/GBS reflecting an acute blood-nerve barrier leakage. In contrast, MS is characterized by an increased intrathecal apoD release as measured by the apoD index. Thus, the results of our study provide the first evidence of an increased intrathecal production of apoD in MS. Moreover, we demonstrate that mean apoD indices are highest in MS patients at the time of their first clinical exacerbation. CSF apoD levels and apoD indices correlate with MS disease duration but not with disability or age. Finally, we found that corticosteroid treatment resulted in significantly elevated CSF apoD levels.

Apolipoprotein E allelic influence on human cerebrospinal fluid apolipoproteins.

The major apolipoproteins (apo) on human cerebrospinal fluid lipoproteins are apoA-I and apoE. Given the association between inheritance of the varepsilon4 allele of the apoE gene (APOE4) and increased susceptibility to Alzheimer's disease, we tested the hypothesis that cerebrospinal fluid apolipoproteins may be influenced by APOE genotype and Alzheimer's disease. Lipoprotein fractions (d < 1.210 g/ml) were isolated from cerebrospinal fluid obtained from individuals with different APOE genotypes and with or without pathologically verified Alzheimer's disease. Apolipoproteins were separated by SDS-polyacrylamide gel electrophoresis and identified by silver nitrate staining, Western blotting, and N-terminal amino acid sequencing. Four protein species were detected by silver nitrate staining in subjects with an APOE3 allele: apoA-I, apoE monomer, apoE-apoA-II heterodimer, and apoE homodimer. In APOE4 homozygotes, only apoA-I and apoE monomer were detected. ApoA-II homodimer was demonstrated in all subjects by Western blotting. The relative levels of apoE- and apoA-II-containing apolipoproteins correlated with APOE genotype but were not altered by Alzheimer's disease. In contrast to apoE, no apoA-II immunoreactivity was observed with pathological structures in Alzheimer's disease brain. These differences in cerebrospinal fluid apolipoproteins may influence lipoprotein trafficking and may be an element in the stratification of risk for Alzheimer's disease with APOE genotype.

Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients.

Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimer's disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 +/- 1.58 microg/ml) and patients with other pathologies (3.29 +/- 1.35 microg/ml) compared with those in the CSF of normal subjects (1.15 +/- 0.71 microg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the epsilon4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.

AP30, a differential protein marker for perilymph and cerebrospinal fluid in middle ear fluid, has been purified and identified as human apolipoprotein D.

Using two-dimensional (2-D) gel electrophoresis, human perilymph and cerebrospinal fluid have been shown to be highly enriched for an acidic protein with MR 30,000, we designated it as AP30. The protein exhibits charge heterogeneity, with at least eight isoforms visible between pI 4.5 to 5.5 on 2-D gels. Purification of the protein was carried out by ammonium sulfate precipitation, polybuffer exchanger column chromatofocusing, and acetone fractional precipitation. The resulting preparation also contains eight spots in the acidic area of 2-D gels, and one broad band located at Mr 30,000 by SDS-PAGE. Digestion of AP30 with neuraminidase causes the isoforms to shift to a more basic position and to consolidate into two primary spots, indicating that AP30 is a variably sialylated glycoprotein. Amino acid analysis of AP30 revealed an amino acid content very similar to that of human apolipoprotein D. Attempts to determine the amino acid sequence demonstrated that the N-terminus is blocked. Edman sequencing of two peptide fragments, generated by cyanogen bromide cleavage of AP30, both revealed sequences having 100% identity to human apolipoprotein D. Western blot analysis of AP30 with the antibody against authentic human apolipoprotein D demonstrated a high degree of cross-reactivity. These studies indicate that AP30 from human perilymph and cerebrospinal fluid is a member of the apolipoprotein D family.

Marked increases in concentrations of apolipoprotein in the cerebrospinal fluid of poliovirus-infected macaques: relations between apolipoprotein concentrations and severity of brain injury.

Apolipoproteins in cerebrospinal fluid (CSF) might have important functional roles in the pathophysiology of brain and lipid metabolism in the vascular component. The present study examined apolipoprotein A-I (apo-A-I) and apolipoprotein E (apo-E) levels in CSF and serum from poliovirus-infected macaques. Poliovirus-infected macaques developed motor deficits and were classified into three groups: (1) muscle weakness in one or both legs; (2) partial paralysis in one or both legs; (3) complete paralysis in one or both legs. No motor deficits were evident in the control or sham-treated macaques. Apo-A-I concentrations in CSF were markedly elevated in poliovirus-infected macaques with weakness, partial or complete paralysis, in comparison with either control or sham-treated animals, and were proportional to the severity of motor impairment. Apo-E concentrations in CSF were also significantly elevated in poliovirus-infected macaques with complete paralysis. The magnitude of increase in CSF apo-A-I or apo-E concentrations was also closely associated with the degree of histologic neurological damage and inflammation (lesion scores). However, no changes in serum apo-A-I and apo-E concentrations were observed in the poliovirus-infected macaques compared with control macaques. Furthermore there were no significant correlations apo-A-I or apo-E concentrations between serum and CSF. We hypothesize that the elevation of apo-A-I and apo-E concentrations after poliovirus infection is caused by immune stimulation within the central nervous system (CNS). Measures of CSF apo-A-I and apo-E levels might serve as a useful marker for the severity and/or the range of CNS injury.