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Background: Multiple system atrophy (MSA) is a rare, progressive, neurodegenerative disorder presenting glia pathology. Still, disease etiology and pathophysiology are unknown, but neuro-inflammation and vascular disruption may be contributing factors to the disease progression. Here, we performed an ex vivo deep proteome profiling of the prefrontal cortex of MSA patients to reveal disease-relevant molecular neuropathological processes. Observations were validated in plasma and cerebrospinal fluid (CSF) of novel cross-sectional patient cohorts. Methods: Brains from 45 MSA patients and 30 normal controls (CTRLs) were included. Brain samples were homogenized and trypsinized for peptide formation and analyzed by high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS). Results were supplemented by western blotting, immuno-capture, tissue clearing and 3D imaging, immunohistochemistry and immunofluorescence. Subsequent measurements of glial fibrillary acid protein (GFAP) and neuro-filament light chain (NFL) levels were performed by immunoblotting in plasma of 20 MSA patients and 20 CTRLs. Finally, we performed a proteome profiling of 144 CSF samples from MSA and CTRLs, as well as other parkinsonian disorders. Data were analyzed using relevant parametric and non-parametric two-sample tests or linear regression tests followed by post hoc tests corrected for multiple testing. Additionally, high-throughput bioinformatic analyses were applied. Results: We quantified more than 4,000 proteins across samples and identified 49 differentially expressed proteins with significantly different abundances in MSA patients compared with CTRLs. Pathway analyses showed enrichment of processes related to fibrinolysis and complement cascade activation. Increased fibrinogen subunit β (FGB) protein levels were further verified, and we identified an enriched recognition of FGB by IgGs as well as intra-parenchymal accumulation around blood vessels. We corroborated blood–brain barrier leakage by a significant increase in GFAP and NFL plasma levels in MSA patients that correlated to disease severity and/or duration. Proteome profiling of CSF samples acquired during the disease course, confirmed increased total fibrinogen levels and immune-related components in the soluble fraction of MSA patients. This was also true for the other atypical parkinsonian disorders, dementia with Lewy bodies and progressive supra-nuclear palsy, but not for Parkinson’s disease patients. Conclusion: Our results implicate activation of the fibrinolytic cascade and immune system in the brain as contributing factors in MSA associated with a more severe disease course.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are synucleinopathies characterized by aggregation of α‐synuclein in brain cells. Recent studies have shown that morphological changes in terms of cerebral nerve cell loss and increase in glia cell numbers, the degree of brain atrophy and molecular and epidemiological findings are more severe in MSA than PD. In the present study, we performed a stereological comparison of cerebellar volumes, granule and Purkinje cells in 13 patients diagnosed with MSA [8 MSA‐P (striatonigral subtype) and 5 MSA‐C (olivopontocerebellar subtype)], 12 PD patients, and 15 age‐matched control subjects. Only brains from MSA‐C patients showed a reduction in the total number of Purkinje cells (anterior lobe) whereas both MSA‐P and MSA‐C patients had reduced Purkinje cell volumes (perikaryons and nuclei volume). The cerebellum of both diseases showed a reduction in the white matter volume compared to controls. The number of granule cells was unaffected in both diseases. Analyses of cell type‐specific mRNA expression supported our structural data. This study of the cerebellum is in line with previous findings in the cerebrum and demonstrates that the degree of morphological changes is more pronounced in MSA‐C than MSA‐P and PD. Further, our results support an explicit involvement of cerebellar Purkinje cells and white matter connectivity in MSA‐C > MSA‐P and points to the potential importance of white matter alterations in PD pathology.

Introduction Ubiquitous naturally occurring autoantibodies (nAbs) against alpha-synuclein (α-syn) may play important roles in the pathogenesis of Multiple System Atrophy (MSA) and Parkinson's disease (PD). Recently, we reported reduced high-affinity/avidity anti-α-syn nAbs levels in plasma from MSA and PD patients, along with distinct inter-group immunoglobulin (Ig)G subclass distributions. The extent to which these observations in plasma may reflect corresponding levels in the cerebrospinal fluid (CSF) is unknown. Methods Using competitive and indirect ELISAs, we investigated the affinity/avidity of CSF anti-α-syn nAbs as well as the CSF and plasma distribution of IgG subclasses and IgM nAbs in a cross-sectional cohort of MSA and PD patients. Results Repertoires of high-affinity/avidity anti-α-syn IgG nAbs were reduced in CSF samples from MSA and PD patients compared to controls. Furthermore, anti-α-syn IgM nAb levels were relatively lower in CSF and plasma from MSA patients but were reduced only in plasma from PD patients. Interestingly, anti-α-syn IgG subclasses presented disease-specific profiles both in CSF and plasma. Anti-α-syn IgG1, IgG2 and IgG3 levels were relatively increased in CSF of MSA patients, whereas PD patients showed increased anti-α-syn IgG2 and reduced anti-α-syn IgG4 levels. Conclusions Differences in the plasma/CSF distribution of anti-α-syn nAbs seem to be a common feature of synucleinopathies. Our data add further support to the notion that MSA and PD patients may have compromised immune reactivity towards α-syn. The differing α-syn-specific systemic immunological responses may reflect their specific disease pathophysiologies. These results are encouraging for further investigation of these immunological mechanisms in neurodegenerative diseases.

Background: Multiple system atrophy (MSA) is a sporadic progressive neurodegenerative disorder with adult onset and unknown etiology. Clinically it is characterized by autonomic failure, cerebellar ataxia, parkinsonism, and corticospinal dysfunction in any combination and with varying severity. Objectives and Methods: To establish the extent of involvement of the white matter in the disease, we have used stereology to quantify the total number of neurons and glial cells (oligodendrocytes, astrocytes, and microglia) in the brains from 10 MSA patients and 11 controls. Results: The mean total number of white matter interstitial neurons in the patient brains was 0.5 × 109 (coefficient of variation = standard deviation/mean = 0.37), which was significantly lower than the 1.1 × 109 (0.41) in the control brains (P = .001) and equal to a reduction by ∼50%. The patient brains had a significantly higher number of white matter microglia, 1.5 × 109 (0.47) versus 0.7 × 109 (0.39) microglia in the control subjects (P = .003) and equal to an increase by ∼ 100%. There was no significant difference in mean total numbers of white matter oligodendrocytes and astrocytes between the groups. Conclusions: We found widespread microgliosis without concomitant astrogliosis in brain white matter in MSA patients and demonstrated an absence of significant oligodendrocyte degeneration. The exact role of oligodendrocytes in MSA pathogenesis, including neurodegeneration, remains to be elucidated. © 2017 International Parkinson and Movement Disorder Society

article i nfo Total numbers of neurons, oligodendrocytes, astrocytes, and microglia in the basal ganglia and red nucleus were estimated in brains from 11 patients with multiple system atrophy (MSA) and 11 age- and gender-matched control subjects with unbiased stereological methods. Compared to the control subjects, the MSA patients had a substantially lower number of neurons in the substantia nigra (p = 0.001), putamen (p = 0.001), and globus pallidus (p b 0.001), and, to a lesser extent in the caudate nucleus (p = 0.03). A significantly lower number of oligodendrocytes were only observed in the putamen (p = 0.04) and globus pallidus (p = 0.01). In the MSA brains the total number of astrocytes was significantly higher in the putamen (p = 0.04) and caudate nucleus (p = 0.01). In all examined regions a highernumberof microglia were found inthe MSA brains with thegreatest difference observed inthe otherwise unaffectedrednucleus (p= 0.001).The resultsfromthestereological study were supported by cell marker expression analyses showing increased markers for activated microglia. Our re- sults suggest that microgliosis is a consistent and severe neuropathological feature of MSA, whereas no wide- spread and substantial loss of oligodendrocytes was observed. We have demonstrated significant neuronal loss in the substantia nigra, striatum, and globus pallidus of patients with MSA, while neurons in other basal ganglia nuclei were spared, supporting the region-specific patterns of neuropathological changes in MSA.

ABSTRACT: Aim: To evaluate the diagnostic value of the level of α-synuclein, DJ-1, and Alzheimer's disease markers in the cerebrospinal fluid (CSF) in patients with parkinsonian syndromes. Materials & methods: We measured α-synuclein, DJ-1 and Alzheimer's disease markers by quantitative immunoassays in 122 CSF samples from patients with Parkinson's disease (PD; n = 38), dementia with Lewy bodies (n = 20), multiple-system atrophy (n = 18), progressive supranuclear palsy (n = 28), corticobasal degeneration (n = 7), unspecified atypical parkinsonian disorders (n = 5) and healthy controls (n = 6). Results: The level of α-synuclein was significantly decreased in CSF from PD patients. For the total tau/α-synuclein ratio, we obtained an area under the curve of 0.80 of the receiver operating characteristics curve for the PD diagnosis. With a cut-off of 1.33, the tau/α-synuclein ratio had 93% diagnostic specificity at 75% sensitivity for PD. Conclusion: CSF α-synuclein measurement may be useful in the differential diagnosis of PD versus other parkinsonian syndromes. CSF α-synuclein is found at low levels in the CSF and requires specifically adjusted assays to be measured.

Multiple system atrophy (MSA) is a sporadic progressive neurodegenerative disorder with adult onset and unknown etiology. Clinically it is characterized by autonomic failure, cerebellar ataxia, parkinsonism, and corticospinal dysfunction in any combination and with varying severity.To establish the extent of involvement of the white matter in the disease, we have used stereology to quantify the total number of neurons and glial cells (oligodendrocytes, astrocytes, and microglia) in the brains from 10 MSA patients and 11 controls.The mean total number of white matter interstitial neurons in the patient brains was 0.5 × 10We found widespread microgliosis without concomitant astrogliosis in brain white matter in MSA patients and demonstrated an absence of significant oligodendrocyte degeneration. The exact role of oligodendrocytes in MSA pathogenesis, including neurodegeneration, remains to be elucidated. © 2017 International Parkinson and Movement Disorder Society.

Background Clinical differentiation between parkinsonian syndromes (PS) remains a challenge despite well-established clinical diagnostic criteria. Specific diagnostic biomarkers have yet to be identified, though in recent years, studies have been published on the aid of certain brain related proteins (BRP) in the diagnosing of PS. We investigated the levels of the light subunit of neurofilament triplet protein (NF-L), total tau and phosphorylated tau, amyloid-β 1-42 , and the soluble α- and β-cleaved fragments of amyloid precursor proteins in a cohort of patients with various PS. Methods Seventy-one patients with different PS and cerebellar disorders were included consecutively over 21 months. CSF was collected at inclusion. Clinical follow-up was performed after 16 months (median; range: 9-30). Statistical comparison was performed after follow-up on 53 patients in four subgroups of PS: multiple system atrophy (MSA)( n = 10), progressive supranuclear palsy (PSP)( n = 10), dementia with Lewy bodies (DLB)( n = 11), and Parkinson’s disease (PD)( n = 22), using the non-parametric Kruskal–Wallis test. Results A statistically significant difference was found for NF-L ( p 1–42, ( p = 0.002, lowest values for DLB), and sAPPα and sAPPβ ( p = 0.03 and 0.02, lower values observed for DLB and MSA). Conclusion We demonstrate a potential role for sAPPα and sAPPβ in distinguishing between PS, a finding that needs to be confirmed in future studies of larger cohorts. There is a tendency towards low levels of Aβ 1–42 in DLB patients in our cohort. Further, our results support findings from previous studies, which indicate an ability to separate atypical PS from PD based on levels of NF-L.

To determine the extent of neocortical involvement in multiple system atrophy (MSA), we used design-based stereological methods to estimate the total numbers of neurons, oligodendrocytes, astrocytes, and microglia in the frontal, parietal, temporal, and occipital cortex of brains from 11 patients with MSA and 11 age- and gender-matched control subjects. The stereological data were supported by cell marker expression analyses in tissue samples from the prefrontal cortex. We found significantly fewer neurons in the frontal and parietal cortex of MSA brains compared with control brains. Significantly more astrocytes and microglia were observed in the frontal, parietal, and temporal cortex of MSA brains, whereas no change in the total number of oligodendrocytes was seen in any of the neocortical regions. There were significantly fewer neurons in the frontal cortex of MSA patients with impaired executive function than in patients with normal executive function. Our results indicate that the involvement of the neocortex in MSA is far more widespread and substantial than previously thought. In addition, our results suggest that the increasingly recognized cognitive impairment in MSA may be related to neuronal loss in the frontal cortex.

Aim: The purpose of this study was to establish the influence of centrifugation and protease activity on the cerebrospinal fluid (CSF) concentrations of DJ-1 and hemoglobin. Materials & methods: The concentrations of DJ-1 and hemoglobin were determined in 12 (DJ-1) and six (hemoglobin) pairs of CSF samples, with one sample being stored without centrifugation and the other being centrifuged at 2000 × g before storage. The DJ-1 concentration was also determined in centrifuged and uncentrifuged CSF containing protease inhibitors and compared with values determined in centrifuged and uncentrifuged CSF samples without protease inhibitors. Furthermore, specific protein concentrations were determined in CSF from two groups, each comprising 23 patients with Parkinson’s disease. In one group the CSF was centrifuged at 1300−1800 × g, 4°C, 10 min, and in the other at 2000 × g, 4°C, 10 min. Results: Centrifugation at 2000 × g resulted in significantly lower CSF DJ-1 concentrations compared with no centrifugation and centrifugation at a lower g-force. There was a significant difference in the hemoglobin concentration between centrifuged and uncentrifuged CSF. In all centrifuged samples the hemoglobin concentration was

Cerebrospinal fluid (CSF) is an ideal biological material in which to search for new biomarkers for improved diagnosis of neurological diseases. During a lumbar puncture between 5 and 15 mL of CSF are obtained. Previous studies have assessed the ventriculo-lumbar concentration gradient of a number of specific proteins. In the present study we took a proteomics approach to investigate the possible concentration gradient of a panel of proteins and peptides in the CSF of 16 patients with neurodegenerative diseases. Using two different mass spectrometry techniques, matrix assisted laser desorption ionization time of flight (MALDI-TOF) and surface enhanced laser desorption ionization time of flight (SELDI-TOF), we found that only one of the investigated proteins, apolipoprotein CI, was significantly decreased between the 1st and the 10th mL of CSF. Furthermore, we confirmed previous results showing a significant decrease in albumin concentration from the first to the last CSF aliquots. In conclusion, we found a significant gradient effect for only two of the measured proteins. However, a standardized procedure for CSF collection for diagnostic and research purposes is crucial to allow comparisons of results between patient groups and between laboratories. This is especially important since CSF is usually collected at several centres and variation in sampled CSF due to pre-analytical factors could complicate the interpretation of the results.