Glypican-4 serum levels are associated with cognitive dysfunction and vascular risk factors in Parkinson's disease.

Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer's disease, which is also a co-pathology in Parkinson's disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson's disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia.

Metal dyshomeostasis in the substantia nigra of patients with Parkinson's disease or multiple sclerosis.

Abnormal metal distribution in vulnerable brain regions is involved in the pathogenesis of most neurodegenerative diseases, suggesting common molecular mechanisms of metal dyshomeostasis. This study aimed to compare the intra- and extra-neuronal metal content and the expression of proteins related to metal homeostasis in the substantia nigra (SN) from patients with Parkinson's disease (PD), multiple sclerosis (MS), and control subjects. Metal quantification was performed via ion-beam micro-analysis in neuromelanin-positive neurons and the surrounding tissue. For proteomic analysis, SN tissue lysates were analyzed on a nanoflow chromatography system hyphenated to a hybrid triple-quadrupole time-of-flight mass spectrometer. We found increased amounts of iron in neuromelanin-positive neurons and surrounding tissue in patients with PD and MS compared to controls (4- to 5-fold higher) that, however, also showed large inter-individual variations. Copper content was systematically lower (-2.4-fold) in neuromelanin-positive neurons of PD patients compared with controls, whereas it remained unchanged in MS. Protein-protein interaction (PPI) network analyses revealed clusters related to Fe and Cu homeostasis among PD-deregulated proteins. An enrichment for the term "metal homeostasis" was observed for MS-deregulated proteins. Important deregulated hub proteins included hemopexin and transferrin in PD, and calreticulin and ferredoxin reductase in MS. Our findings show that PD and MS share commonalities in terms of iron accumulation in the SN. Concomitant proteomics experiments revealed PPI networks related to metal homeostasis, substantiating the results of metal quantification.

Brainstem atrophy in dementia with Lewy bodies compared with progressive supranuclear palsy and Parkinson's disease on MRI.

BACKGROUND: Although Dementia with Lewy bodies (DLB) is the second most common form of dementia in elderly patients, it remains underdiagnosed compared with Alzheimer's (AD) and Parkinson's diseases (PD). This may be explained by overlapping clinical symptoms, e.g. Parkinsonism. While current MRI research focuses primarily on atrophy patterns of the frontal and temporal lobes, we focus on brainstem characteristics of DLB. In particular, we focused on brainstem atrophy patterns distinguishing DLB from Progressive Supranuclear Palsy (PSP) and PD based as the most common differential diagnoses. METHODS: We identified patients diagnosed with DLB, PD, PSP, and a control group (CTRL) in our psychiatric and neurological archives. All patients with competing diagnoses and without a high-quality T1 MPRAGE 3D dataset were excluded. We assessed atrophy patterns in all patients (1) manually and (2) using FastSurfer's segmentation algorithm in combination with FreeSurfer's brainstem volumetric calculations. We compared classical measurement methods and ratios with automated volumetric approaches. RESULTS: One hundred two patients were enrolled and evaluated in this study. Patients with DLB (n = 37) showed on average less atrophy of the brainstem than patients with PSP (n = 21), but a significantly more pronounced atrophy than patients with PD (n = 36) and the control group (CTRL, n = 8). The mean measured sagittal diameters of the midbrain were 8.17 ± 1.06 mm (mean ± standard deviation) for PSP, 9.45 ± 0.95 mm for DLB, 10.37 ± 0.99 mm for PD and 10.74 ± 0.70 for CTRL. The mean measured areas of the midbrain were 81 ± 18 mm2 for PSP, 105 ± 17 mm2 for DLB, 130 ± 26 mm2 for PD and 135 ± 23 mm2 for CTRL. The mean segmented volumes of the midbrain were 5595 ± 680 mm3 for PSP, 6051 ± 566 mm3 for DLB, 6646 ± 802 mm3 for PD and 6882 ± 844 mm3 for CTRL. The calculated midbrain pons ratios did not show superiority over the absolute measurements of the midbrain for distinguishing PSP from DLB. Because of the relatively uniform atrophy throughout the brainstem, the ratios were not suitable for distinguishing DLB from PD. CONCLUSIONS: DLB patients exhibit homogenous atrophy of the brainstem and can be distinguished from patients with PSP and PD by both manual measurement methods and automated volume segmentation using absolute values or ratios.

Prospective CERAD Neuropsychological Assessment in Patients With Multiple System Atrophy.

The objective of the study was to characterize the pattern of cognitive dysfunction in patients with multiple system atrophy (MSA) applying a standardized neuropsychological assessment. A total of 20 patients with the diagnosis of probable or possible MSA were enrolled for neuropsychological assessment applying the CERAD plus battery. All patients were tested at baseline and 14/20 patients received additional follow-up assessments (median follow-up of 24 months). Additionally, relationship between cortical thickness values/subcortical gray matter volumes and CERAD subitems was evaluated at baseline in a subgroup of 13/20 patients. Trail Making Test (TMT) was the most sensitive CERAD item at baseline with abnormal performance (z-score < -1.28) in one or both pathological TMT items (TMT-A, TMT-B) in 60% of patients with MSA. Additionally, there was a significant inverse correlation between the volume of the left and the right accumbens area and the TMT A item after adjusting for age (left side: p = 0.0009; right side p = 0.003). Comparing both subtypes, patients with MSA-C had significant lower values in phonemic verbal fluency (p = 0.04) and a trend for lower values in semantic verbal fluency (p = 0.06) compared to MSA-P. Additionally, patients with MSA-C showed significantly worse performance in the TMT-B task (p = 0.04) and a trend for worse performance in the TMT-A task (p = 0.06). Concerning longitudinal follow-up, a significant worsening in the TMT-B (p = 0.03) can be reported in MSA. In conclusion, frontal-executive dysfunction presents the hallmark of cognitive impairment in MSA.

The relevance of synuclein autoantibodies as a biomarker for Parkinson's disease.

Several studies have investigated if the levels of α-synuclein autoantibodies (α-syn AAb) differ in serum of Parkinson's disease (PD) patients and healthy subjects. Reproducible differences in their levels could serve as a biomarker for PD. The results of previous studies however remain inconclusive. With the largest sample size examined so far, we aimed to validate serum α-syn AAb levels as a biomarker for PD and investigated the presence of AAbs against other synucleins. We performed ELISA and immunoblots to determine synuclein AAb levels in the serum of 295 subjects comprising 157 PD patients from two independent cohorts, 46 healthy subjects, and 92 patients with other neurodegenerative disorders. Although serum α- and ß-syn AAb levels were significantly reduced in patients with PD and other neurodegenerative disorders as compared to controls, the AAb levels displayed high inter-and intra-cohort variability. Furthermore, α-syn AAb levels showed no correlation to clinical parameters like age, disease duration, disease severity, and gender, that might also be directed against beta- and gamma-syn. In conclusion, serum synuclein AAb levels do allow the separation of PD from healthy subjects but not from other neurodegenerative disorders. Thus, synuclein AAbs cannot be regarded as a reliable biomarker for PD.

Validation of Plasma and CSF Neurofilament Light Chain as an Early Marker for Sporadic Creutzfeldt-Jakob Disease.

Biomarkers are becoming increasingly important for the differential diagnosis of neurodegenerative diseases. Previous observations indicated neurofilament light chain (NfL) as a potential blood-based biomarker for sporadic Creutzfeldt-Jakob disease (sCJD). Here, we investigated the stability, inter-assay/intra-assay variation and the regulation of NfL levels in CSF and plasma in a large cohort of sCJD patients by using a single-molecule array (SIMOA). We defined cutoffs for an accurate diagnosis and measured plasma NfL level in prion-infected mice models at different time points to identify the potential dynamics throughout the disease. Our analyses confirmed CSF and plasma NfL as stable and consistent marker for sCJD. Receiver operating characteristic (ROC) curve analysis showed an AUC of 0.92-0.93 to distinguish sCJD from control groups. Newly defined cutoffs revealed good diagnostic accuracies of CSF and plasma NfL, indicated by a sensitivity of 80-83.5% and a specificity of 87.4-91%. Studies on two humanized prion-infected mice lines (Tg340-PRNP 129MM and Tg361-PRNP 129VV) revealed increased plasma NfL levels in a late pre-clinical or very early clinical stage between 120-150 days post-inoculation. In conclusion, our work supports the potential use of CSF and plasma NfL as a very early biomarker in sCJD diagnostic with good diagnostic accuracies.

Baseline Cerebrospinal Fluid α-Synuclein in Parkinson's Disease Is Associated with Disease Progression and Cognitive Decline.

Biomarkers are increasingly recognized as tools in the diagnosis and prognosis of neurodegenerative diseases. No fluid biomarker for Parkinson's disease (PD) has been established to date, but α-synuclein, a major component of Lewy bodies in PD and dementia with Lewy bodies (DLB), has become a promising candidate. Here, we investigated CSF α-synuclein in patients with PD (n = 28), PDD (n = 8), and DLB (n = 5), applying an electrochemiluminescence immunoassay. Median values were non-significantly (p = 0.430) higher in patients with PDD and DLB (287 pg/mL) than in PD (236 pg/mL). A group of n = 36 primarily non-demented patients with PD and PDD was clinically followed for up to two years. A higher baseline α-synuclein was associated with increases in Hoehn and Yahr classifications (p = 0.019) and Beck Depression Inventory scores (p < 0.001) as well as worse performance in Trail Making Test A (p = 0.017), Trail Making Test B (p = 0.043), and the Boston Naming Test (p = 0.002) at follow-up. Surprisingly, higher levels were associated with a better performance in semantic verbal fluency tests (p = 0.046). In summary, CSF α-synuclein may be a potential prognostic marker for disease progression, affective symptoms, and executive cognitive function in PD. Larger-scaled studies have to validate these findings and the discordant results for single cognitive tests in this exploratory investigation.

Multi-omic landscaping of human midbrains identifies disease-relevant molecular targets and pathways in advanced-stage Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder whose prevalence is rapidly increasing worldwide. The molecular mechanisms underpinning the pathophysiology of sporadic PD remain incompletely understood. Therefore, causative therapies are still elusive. To obtain a more integrative view of disease-mediated alterations, we investigated the molecular landscape of PD in human post-mortem midbrains, a region that is highly affected during the disease process. METHODS: Tissue from 19 PD patients and 12 controls were obtained from the Parkinson's UK Brain Bank and subjected to multi-omic analyses: small and total RNA sequencing was performed on an Illumina's HiSeq4000, while proteomics experiments were performed in a hybrid triple quadrupole-time of flight mass spectrometer (TripleTOF5600+) following quantitative sequential window acquisition of all theoretical mass spectra. Differential expression analyses were performed with customized frameworks based on DESeq2 (for RNA sequencing) and with Perseus v.1.5.6.0 (for proteomics). Custom pipelines in R were used for integrative studies. RESULTS: Our analyses revealed multiple deregulated molecular targets linked to known disease mechanisms in PD as well as to novel processes. We have identified and experimentally validated (quantitative real-time polymerase chain reaction/western blotting) several PD-deregulated molecular candidates, including miR-539-3p, miR-376a-5p, miR-218-5p and miR-369-3p, the valid miRNA-mRNA interacting pairs miR-218-5p/RAB6C and miR-369-3p/GTF2H3, as well as multiple proteins, such as CHI3L1, HSPA1B, FNIP2 and TH. Vertical integration of multi-omic analyses allowed validating disease-mediated alterations across different molecular layers. Next to the identification of individual molecular targets in all explored omics layers, functional annotation of differentially expressed molecules showed an enrichment of pathways related to neuroinflammation, mitochondrial dysfunction and defects in synaptic function. CONCLUSIONS: This comprehensive assessment of PD-affected and control human midbrains revealed multiple molecular targets and networks that are relevant to the disease mechanism of advanced PD. The integrative analyses of multiple omics layers underscore the importance of neuroinflammation, immune response activation, mitochondrial and synaptic dysfunction as putative therapeutic targets for advanced PD.

Detection of Cerebrospinal Fluid Neurofilament Light Chain as a Marker for Alpha-Synucleinopathies.

Alpha-synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are a class of neurodegenerative diseases. A diagnosis may be challenging because clinical symptoms partially overlap, and there is currently no reliable diagnostic test available. Therefore, we aimed to identify a suitable marker protein in cerebrospinal fluid (CSF) to distinguish either between different types of alpha-synucleinopathies or between alpha-synucleinopathies and controls. In this study, the regulation of different marker protein candidates, such as alpha-synuclein (a-Syn), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and total tau (tau) in different types of alpha-synucleinopathies, had been analyzed by using an ultrasensitive test system called single-molecule array (SIMOA). Interestingly, we observed that CSF-NfL was significantly elevated in patients with DLB and MSA compared to patients with PD or control donors. To differentiate between groups, receiver operating characteristic (ROC) curve analysis resulted in a very good diagnostic accuracy as indicated by the area under the curve (AUC) values of 0.87-0.92 for CSF-NfL. Furthermore, we observed that GFAP and tau were slightly increased either in DLB or MSA, while a-Syn levels remained unregulated. Our study suggests NfL as a promising marker to discriminate between different types of alpha-synucleinopathies or between DLB/MSA and controls.

Cerebrospinal Fluid Iron-Ferritin Ratio as a Potential Progression Marker for Parkinson's Disease.

Longitudinal PD CSF samples were subjected to ICP-MS and the total amount of iron and other bioelements was quantified. Additionally, ferritin and protein biomarkers of neurodegeneration were measured. Over time, mean iron levels significantly increased while levels of ferritin decreased.

Mild Amnestic Cognitive Impairment and Depressive Symptoms in Autoimmune Encephalitis Associated with Serum Anti-Neurexin-3α Autoantibodies.

(1) Background: autoimmune encephalitis associated with neurexin-3α antibodies is a seldom reported disease entity often accompanied by a severe clinical neuropsychiatric syndrome. (2) Method: we report on the case of a 58-year-old man diagnosed with neurexin-3α-associated autoimmune encephalitis revealing cognitive decline and depression before the proof of neurexin-3α antibodies. He underwent neuropsychological testing, peripheral blood and cerebrospinal fluid analysis, neuroimaging and electroencephalography. (3) Results: our patient's main clinical feature was amnestic cognitive decline in combination with depressive symptoms. CSF analysis showed elevated phosphorylated tau protein 181 and positive proof of serum neurexin-3α antibodies in a cell-based assay. An 18F-FDG-PET/CT of the brain initially showed bilateral cerebral hypometabolism prefrontal and parietal, which was absent in follow up. The brain MRI was unremarkable. EEG recordings showed frontotemporal slowing in the theta and delta range. (4) Conclusions: taken together, we assumed autoimmune encephalitis associated with serum neurexin-3α antibodies. To the best of our knowledge, we are the first to report on a predominantly mild clinical manifestation entailing amnestic mild cognitive impairment in addition to depression, thus broadening the clinical spectrum associated with neurexin-3α antibodies.

Case Report: Findings Suggestive of Paraclinical Progressive Multifocal Leukoencephalopathy and Lung Cancer-Derived Brain Metastases in an MS Patient Treated With Fingolimod.

Fingolimod represents a highly effective disease-modifying drug in patients with active relapsing-remitting multiple sclerosis (RRMS). Its immunosuppressive effects can mediate adverse events like increased risk of cancer development or appearance of opportunistic infections. Progressive multifocal leukoencephalopathy (PML)-representing a severe opportunistic infection-has been only infrequently described during Fingolimod treatment. Here, we present a case of a 63-year-old women with pre-diagnosed RRMS who presented with new multiple cerebral lesions in a routine MRI scan, also including a tumefactive lesion in the left parietal lobe, eventually leading to the diagnosis of brain metastases derived by an adenocarcinoma of the lung. Additionally, a JCV-DNA-PCR in the cerebrospinal fluid revealed positive results, corresponding to a paraclinical progressive multifocal leukoencephalopathy. In conclusion, adverse events potentially associated with immunosuppression can occur during Fingolimod treatment. In this context, the occurrence of cancer and opportunistic infections should be carefully monitored. Here, we report a case in which JCV-DNA-PCR in the cerebrospinal fluid suggests asymptomatic PML and simultaneously lung cancer brain metastases developed. While it is rather unlikely that either event occurred as an adverse event of fingolimod treatment, a contributing effect cannot be formally excluded.

Human cerebrospinal fluid data for use as spectral library, for biomarker research.

Spectral libraries generated by data dependent acquisition (DDA) are a useful tool for the analysis of data created by data independent acquisition (DIA) in mass spectrometry. The quality of DIA analysis is dependent on the quality of the spectral library. We used cerebrospinal fluid (CSF) of patients with Parkinson's disease and healthy controls to create a spectral library of human CSF proteome. To this date, there is no validated CSF biomarker for Parkinson's disease. This data set may therefore be valuable for the future analysis of CSF proteins. Part of the samples consisted of fractions that were separated by gel electrophoresis. After tryptic digestion, all samples were spiked with indexed retention time (iRT) peptides and were measured using a DDA mass spectrometry approach. The here provided data set can be used as a CSF-specific spectral library. Data files generated from the described workflow are hosted in the public repository ProteomeXchange under the identifier PXD013487.

Increased alpha-synuclein tear fluid levels in patients with Parkinson's disease.

The objective of the study was to estimate if altered levels of alpha-synuclein can be detected in tear fluid of patients with Parkinson's disease (PD). Therefore, tear fluid samples of 75 PD patients, 75 control subjects and 31 atypical Parkinsonian patients were collected and analyzed in triplicates using an ultra-sensitive single molecule array (SIMOA) system and applying a human alpha-synuclein immunoassay. In PD, levels of total soluble alpha-synuclein were significantly increased compared to control subjects (p = 0.03; AUC PD vs. controls 0.60). There was no difference comparing PD patients stratified by Hoehn & Yahr stages and atypical Parkinsonian syndromes stratified by tauopathies and non-PD-synucleinopathies against each other (p > 0.05). In conclusion, alpha-synuclein can be detected and quantified in tear fluid, revealing small but significant differences in total alpha-synuclein levels between PD and control subjects. Tear fluid can be collected non-invasively and risk-free, therefore presenting a promising source for further biomarker research.

Compassionate Use of the ROCK Inhibitor Fasudil in Three Patients With Amyotrophic Lateral Sclerosis.

The Rho kinase (ROCK) inhibitor Fasudil is a promising drug for a disease-modifying therapy of amyotrophic lateral sclerosis (ALS). In preclinical models, Fasudil was shown to increase motor neuron survival, inhibit axonal degeneration, enhance axonal regeneration and modulate microglial function in vitro and in vivo. It prolonged survival and improved motor function of SOD1-G93A-mice. Recently, a phase IIa clinical trial has been commenced to investigate the safety, tolerability, and efficacy of Fasudil in ALS patients at an early stage of disease (ROCK-ALS trial, NCT03792490, Eudra-CT-Nr.: 2017-003676-31). Although Fasudil has been approved in Japan for many years for the treatment of vasospasms following subarachnoid hemorrhage and is known to have a favorable side effect profile in these patients, there is no data on its use in human patients with ALS or any other neurodegenerative conditions. Here, we report the first three cases of compassionate use of Fasudil in patients with ALS. Between May 2017 and February 2019, one male (66 years old) and two female (62 and 68 years old) subjects with probable or definite ALS according to the El Escorial criteria (one of the females having a pathogenic SOD1 mutation) were administered Fasudil 30 mg intravenously twice daily over 45 min on 20 consecutive working days. Blood pressure, heart rate and routine laboratory tests were constantly controlled. All three subjects tolerated the Fasudil infusions well without any obvious side effects. Interestingly, the slow vital capacity showed a significant increase in one of the patients. Taken together, we report here the first compassionate use of the ROCK inhibitor Fasudil in three ALS patients, which was well-tolerated.

Chromogranin A Analysis in the Differential Diagnosis Across Lewy Body Disorders.

BACKGROUND: Chromogranin A (CgA) is a general marker of gut endocrine cells, which are part of the "gut-brain axis" in Parkinson's disease (PD). OBJECTIVE: We analyzed CgA as a marker of synaptic dysfunction to assess its role in the differential diagnosis across different Lewy body disorders. METHODS: We analyzed the CgA levels in the cerebrospinal fluid (CSF) and serum from 54 patients covering the spectrum of Lewy body disorders [13 Parkinson's disease (PD), 17 Parkinson's disease dementia (PDD), 24 dementia with Lewy bodies (DLB)] and 14 controls using an ELISA. RESULTS: A positive correlation was noted between CSF and serum CgA levels (ρ= 0.47, 95% CI: 0.24 to 0.65, p < 0.0001). The highest values of CgA in CSF and in serum were measured in DLB and there was a significant difference between DLB and PDD (p = 0.03 and p = 0.004). The serum levels of CgA in controls achieved lower values compared to DLB (p = 0.006). There was a gradual increase in serum levels from PD to PDD and DLB. An inverse correlation was seen between the CSF level of CgA and Aß42 (ρ  = -0.296, 95% CI: -0.51 to -0.04, p = 0.02). CONCLUSION: The incorporation of CgA analysis as an additional biomarker may be useful in the diagnostic work-up of Lewy body dementia. CgA analysis may be relevant in distinguishing DLB from PDD patients and presumably early stages of PD. Our data on altered serum levels in DLB pave the way to the development of blood-based parameters for the differential diagnosis, which however needs to be confirmed in a prospective study.