Identification of cerebrospinal fluid biomarkers for parkinsonism using a proteomics approach.

The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson's disease (PD, n = 10), multiple system atrophy patients (MSA, n = 5) and non-neurological controls (n = 10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n = 46), atypical parkinsonism patients (AP; MSA, n = 17; Progressive supranuclear palsy; n = 8) and non-neurological controls (n = 39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that the discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.

Cerebrospinal Fluid Galectin-1 Levels Discriminate Patients with Parkinsonism from Controls.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in elderly people. Currently, the diagnosis of PD is based on neurological examination, neuroimaging, and the response to dopaminergic medication. The diagnosis can be challenging, especially at early disease stages, when the symptoms of patients with atypical parkinsonism (APD) may strongly overlap. Therefore, reliable biomarkers that are able to identify patients with PD are much needed. Here, we aimed to identify and validate new biomarkers for PD in cerebrospinal fluid (CSF). We performed a profiling experiment using mass spectrometry (MS) of CSF from ten PD patients and ten matched non-neurological controls. We selected one protein, galectin-1 (Gal-1), which was differentially expressed in PD vs. controls, and quantified its concentrations in CSF by enzyme-linked immunosorbent assay (ELISA) in three new cohorts of 37 PD patients, 21 APD patients, and 44 controls. CSF levels of Gal-1 were lower in PD in both the discovery and validation experiments and discriminated PD from controls with moderate-high accuracy levels (ELISA: area under the curve = 0.7). Similar levels of Gal-1 were found in PD and APD. Gal-1 levels were correlated to age in all groups and correlated in the PD patients to CSF levels of total tau, phosphorylated tau, neurofilament light chain (NFL), and the mini-mental state examination (MMSE) score. We conclude that MS profiling of proteins may be a useful tool to identify novel biomarkers of neurological diseases and that CSF Gal-1 levels may discriminate PD from non-neurological controls.

Regulator of oligodendrocyte maturation, miR-219, a potential biomarker for MS.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating and degenerative disease of the central nervous system. Normally, demyelination is followed by remyelination, which requires repopulation of a demyelinated area by oligodendrocyte precursor cells. Although large numbers of precursor cells are present in MS lesions, remyelination often fails, in part by the inability of precursor cells to differentiate into mature myelin-forming cells. In mouse and rat, miR-219 is required for this differentiation. Previously, we identified decreased miR-219 expression in tissue of MS patients compared to controls. Cell-free miRNAs have been detected in many different body fluids including cerebrospinal fluid (CSF) and may reflect disease processes going on in the central nervous system. This prompted us to investigate the biomarker performance of CSF miR-219 for MS diagnosis. METHODS: Quantitative PCR was performed measuring miR-219 levels in CSF of MS patients and controls in three independent cohorts. RESULTS: All three cohorts of MS patients and controls revealed that absence of miR-219 detection in CSF is consistently associated with MS. CONCLUSIONS: We have been able to identify and validate absence of miR-219 detection in CSF of MS patients compared to controls, suggesting that it may emerge as a candidate biomarker for MS diagnosis.

MicroRNAs in Cerebrospinal Fluid as Potential Biomarkers for Parkinson's Disease and Multiple System Atrophy.

Parkinson's disease (PD) and multiple system atrophy (MSA) are both part of the spectrum of neurodegenerative movement disorders and α-synucleinopathies with overlap of symptoms especially at early stages of the disease but with distinct disease progression and responses to dopaminergic treatment. Therefore, having biomarkers that specifically classify patients, which could discriminate PD from MSA, would be very useful. MicroRNAs (miRNAs) regulate protein translation and are observed in biological fluids, including cerebrospinal fluid (CSF), and may therefore have potential as biomarkers of disease. The aim of our study was to determine if miRNAs in CSF could be used as biomarkers for either PD or MSA. Using quantitative PCR (qPCR), we evaluated expression levels of 10 miRNAs in CSF patient samples from PD (n = 28), MSA (n = 17), and non-neurological controls (n = 28). We identified two miRNAs (miR-24 and miR-205) that distinguished PD from controls and four miRNAs that differentiated MSA from controls (miR-19a, miR-19b, miR-24, and miR-34c). Combinations of miRNAs accurately discriminated either PD (area under the curve (AUC) = 0.96) or MSA (AUC = 0.86) from controls. In MSA, we also observed that miR-24 and miR-148b correlated with cerebellar ataxia symptoms, suggesting that these miRNAs are involved in cerebellar degeneration in MSA. Our findings support the potential of miRNA panels as biomarkers for movement disorders and may provide more insights into the pathological mechanisms related to these disorders.

MicroRNA-29a Is a Candidate Biomarker for Alzheimer's Disease in Cell-Free Cerebrospinal Fluid.

The identification of reliable biomarkers for Alzheimer's disease (AD) remains challenging. Recently, abnormal levels of microRNAs (miRNAs) miR-27a, miR-29a, miR-29b, and miR-125b in cerebrospinal fluid (CSF) of AD patients were reported. We aimed to confirm the biomarker potential of these miRNAs for AD diagnosis. Additionally, we examined the influence of blood contamination on CSF miRNA levels as potential confounding factor. We studied expression levels of the four miRNAs by quantitative PCR in CSF samples of AD patients and non-demented controls, and in blood-spiked CSF. Levels of miR-29a, but not of the other three miRNAs, were increased by a factor of 2.2 in CSF of AD patients. Spiking of small amounts of blood into CSF revealed that miR-27a and miR-29a, but not miR-125b levels were strongly influenced by the number of blood cells in the sample. In conclusion, miR-29a may be a candidate biomarker for AD, but only when used in cell-free CSF.

Inhibition of α-synuclein aggregation by small heat shock proteins.

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.

Small heat shock proteins induce a cerebral inflammatory reaction.

More than 80% of Alzheimer's disease (AD) patients have some degree of cerebral amyloid angiopathy (CAA). In addition to arteries and veins, capillaries can also be affected. Capillary CAA (capCAA), rather than CAA in larger vessels, is associated with flame-like amyloid-beta (Aß) deposits that may extend beyond the vessel wall and radiate into the neuropil, a phenomenon also known as "dyshoric angiopathy." Aß deposits in AD, parenchymal as well as (cap)CAA and dyshoric angiopathy, are associated with a local inflammatory reaction, including activation of microglial cells and astrocytes that, among others, produce cytokines and reactive oxygen species. This neuroinflammatory reaction may account for at least part of the cognitive decline. In previous studies we observed that small heat shock proteins (sHsps) are associated with Aß deposits in AD. In this study the molecular chaperones Hsp20, HspB8 and HspB2B3 were found to colocalize with CAA and capCAA in AD brains. In addition, Hsp20, HspB8 and HspB2B3 colocalized with intercellular adhesion molecule 1 (ICAM-1) in capCAA-associated dyshoric angiopathy. Furthermore, we demonstrated that Hsp20, HspB8 and HspB2B3 induced production of interleukin 8, soluble ICAM-1 and monocyte chemoattractant protein 1 by human leptomeningeal smooth muscle cells and human brain astrocytes in vitro and that Hsp27 inhibited production of transforming growth factor beta 1 and CD40 ligand. Our results suggest a central role for sHsps in the neuroinflammatory reaction in AD and CAA and thus in contributing to cognitive decline.

Sulfation of heparan sulfate associated with amyloid-beta plaques in patients with Alzheimer's disease.

Alzheimer's disease (AD) is characterized by pathological lesions such as amyloid-beta (Abeta) plaques and cerebral amyloid angiopathy. Both these lesions consist mainly of aggregated Abeta protein and this aggregation is affected by macromolecules such as heparan sulfate (HS) proteoglycans. Previous studies demonstrated that HS enhances fibrillogenesis of Abeta and that this enhancement is dependent on the degree of sulfation of HS. In addition, it has been reported that these sulfation epitopes do not occur randomly but have a defined tissue distribution. Until now, the distribution of sulfation epitopes of HS has not yet been studied in human brain. We investigated whether a specific HS epitope is associated with Abeta plaques by performing immunohistochemistry on occipital neocortical and hippocampal tissue sections from AD patients using five HS epitope-specific phage display antibodies. Antibodies recognizing highly N-sulfated HS demonstrated the highest level of staining in both fibrillar Abeta plaques and non-fibrillar Abeta plaques, whereas antibodies recognizing HS regions with a lower degree of N-sulfate modifications were only immunoreactive with fibrillar Abeta plaques. Thus, our results suggest that a larger variety of HS epitopes is associated with fibrillar Abeta plaques, but the HS epitopes associated with non-fibrillar Abeta plaques seem to be more restricted, selectively consisting of highly N-sulfated epitopes.

Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Abeta40 (DAbeta(1-40)) are modulated by sulfate moieties of heparin.

Glycosaminoglycans (GAGs), in particular as part of heparan sulfate proteoglycans, are associated with cerebral amyloid angiopathy (CAA). Similarly, GAGs are also associated with the severe CAA found in patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), where the amyloid beta (Abeta) peptide contains the Dutch mutation (DAbeta(1-40)). This suggests a role for GAGs in vascular Abeta aggregation. It was the aim of this study to investigate the effect of different GAGs (heparin, chondroitin sulfate, heparan sulfate), the macromolecule dextran sulfate and, using desulfated heparins, the role of GAG sulfate moieties on the in vitro aggregation of CAA-associated DAbeta(1-40) and on DAbeta(1-40)-induced toxicity of cultured cerebrovascular cells. We also aimed to study the in vivo distribution of various sulfated heparan sulfate GAG epitopes in CAA. Of all GAGs tested, heparin was the strongest inducer of aggregation of DAbeta(1-40) in the different aggregation assays, with both heparin and heparan sulfate reducing Abeta-induced cellular toxicity. Furthermore, (partial) removal of the sulfate moieties of heparin partially abolished the effects of heparin on aggregation and cellular toxicity, suggesting an essential role for the sulfate moieties in heparin. Finally, we demonstrated the in vivo association of sulfated heparan sulfate (HS) GAGs with CAA. We conclude that sulfate moieties within GAGs, like heparin and HS, have an important role in Abeta aggregation in CAA and in Abeta-mediated toxicity of cerebrovascular cells.

Apolipoprotein E protects cultured pericytes and astrocytes from D-Abeta(1-40)-mediated cell death.

Cerebral amyloid angiopathy (CAA) is a common pathological finding in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type; in this latter condition it is caused by deposition of mutated amyloid beta protein (Abeta Glu22Gln; D-Abeta(1-40)). Previously, we found a dependence of the Abeta-mediated toxicity and apolipoprotein E (apoE) production by cultured pericytes on apoE genotype. Given their close association with the cerebrovascular wall both astrocytes and pericytes may be involved in CAA development, a process that includes Abeta deposition and clearance and that may be affected by interaction with locally produced apolipoprotein E (apoE). Although astrocytes are regarded as the major source of apolipoprotein E (apoE) in the brain, also pericytes produce apoE. In this study we compared the apoE production capacity, the effects of apoE on D-Abeta(1-40) internalization, D-Abeta(1-40) cell surface accumulation and the vulnerability for D-Abeta(1-40)-induced toxicity of either cell type in order to quantify the relative contributions of astrocytes and pericytes in the various processes that contribute to CAA formation. Strikingly, cultured astrocytes produced only 3-10% of the apoE amounts produced by pericytes. Furthermore, pericytes with the apoE epsilon4 allele produced three times less apoE and were more vulnerable to D-Abeta(1-40) treatment than pericytes without an epsilon4 allele. Such relations were not observed with astrocytes in vitro. Both pericytes and astrocytes, however, were protected from Abeta-induced cytotoxicity by high levels of pericyte-derived apoE, but not recombinant apoE. In addition, pericyte-derived apoE dose-dependently decreased both internalization of Abeta and Abeta accumulation at the cell surface in either cell type. The present data suggest that apoE produced by pericytes, rather than astrocyte-produced apoE, modulates Abeta cytotoxicity and Abeta removal near the vasculature in the brain. Furthermore, since apoE production in pericytes is genotype dependent, this may contribute to the apoE genotype-dependent development of CAA in vivo.