Assessment of immunoprecipitation with subsequent immunoassays for the blood-based diagnosis of Alzheimer's disease.

The Aß42/40 ratio and the concentration of phosphorylated Tau181 in blood plasma represent attractive biomarkers for Alzheimer's disease. As a means for reducing potential matrix effects, which may interfere with plasma immunoassays, we have previously developed a pre-analytical sample workup by semi-automated immunoprecipitation. Here we test the compatibility of pre-analytical immunoprecipitations with automated Aß1-40, Aß1-42 and phosphorylated Tau181 immunoassays on the Lumipulse platform and compare the diagnostic performance of the respective immunoprecipitation immunoassay approaches with direct plasma measurements. 71 participants were dichotomized according to their Aß42/40 ratios in cerebrospinal fluid into the diagnostic groups amyloid-positive (n = 32) and amyloid-negative (n = 39). The plasma Aß1-42/1-40 ratio and phosphorylated Tau181 levels were determined on the Lumipulse G600II platform (Fujirebio) by direct measurements in EDTA-plasma or after Aß- or Tau-immunoprecipitation, respectively. Pre-analytical immunoprecipitation of Aß turned out to be compatible with the Lumipulse Aß assays and resulted in a numerical, yet statistically not significant increase in the area under the ROC curve for plasma Aß1-42/1-40. Additionally, we observed a significant increase in the standardised effect size (Cohen's D). Pre-analytical immunoprecipitation of Tau resulted in increased differences between the diagnostic groups in terms of median and mean phosphorylated Tau 181 levels. Furthermore, we observed a greater Cohen's d (p < 0.001) and a larger area under the ROC curve (p = 0.038) after Tau-IP. Our preliminary findings in a small, preselected sample indicate that pre-analytical immunoprecipitation may have the potential to improve the diagnostic performance of plasma biomarker immunoassays for Aß1-42/1-40 and phosphorylated Tau181 to predict brain amyloid deposition.

Inflammatory CSF profiles and longitudinal development of cognitive decline in sporadic and GBA-associated PD.

Inflammation modifies the incidence and progression of Parkinson's disease (PD). By using 30 inflammatory markers in CSF in 498 people with PD and 67 people with dementia with Lewy bodies (DLB) we show that: (1) levels of ICAM-1, Interleukin-8, MCP-1, MIP-1 beta, SCF and VEGF were associated with clinical scores and neurodegenerative CSF biomarkers (Aß1-42, t-Tau, p181-Tau, NFL and α-synuclein). (2) PD patients with GBA mutations show similar levels of inflammatory markers compared to PD patients without GBA mutations, even when stratified by mutation severity. (3) PD patients who longitudinally developed cognitive impairment during the study had higher levels of TNF-alpha at baseline compared to patients without the development of cognitive impairment. (4) Higher levels of VEGF and MIP-1 beta were associated with a longer duration until the development of cognitive impairment. We conclude that the majority of inflammatory markers is limited in robustly predicting longitudinal trajectories of developing cognitive impairment.

TDP-43 CSF Concentrations Increase Exponentially with Age in Metropolitan Mexico City Young Urbanites Highly Exposed to PM2.5 and Ultrafine Particles and Historically Showing Alzheimer and Parkinson's Hallmarks. Brain TDP-43 Pathology in MMC Residents Is Associated with High Cisternal CSF TDP-43 Concentrations.

Environmental exposures to fine particulate matter (PM2.5) and ultrafine particle matter (UFPM) are associated with overlapping Alzheimer's, Parkinson's and TAR DNA-binding protein 43 (TDP-43) hallmark protein pathologies in young Metropolitan Mexico City (MMC) urbanites. We measured CSF concentrations of TDP-43 in 194 urban residents, including 92 MMC children aged 10.2 ± 4.7 y exposed to PM2.5 levels above the USEPA annual standard and to high UFPM and 26 low pollution controls (11.5 ± 4.4 y); 43 MMC adults (42.3 ± 15.9 y) and 14 low pollution adult controls (33.1 ± 12.0 y); and 19 amyotrophic lateral sclerosis (ALS) patients (52.4 ± 14.1 y). TDP-43 neuropathology and cisternal CSF data from 20 subjects­15 MMC (41.1 ± 18.9 y) and 5 low pollution controls (46 ± 16.01 y)­were included. CSF TDP-43 exponentially increased with age (p < 0.0001) and it was higher for MMC residents. TDP-43 cisternal CSF levels of 572 ± 208 pg/mL in 6/15 MMC autopsy cases forecasted TDP-43 in the olfactory bulb, medulla and pons, reticular formation and motor nuclei neurons. A 16 y old with TDP-43 cisternal levels of 1030 pg/mL exhibited TDP-43 pathology and all 15 MMC autopsy cases exhibited AD and PD hallmarks. Overlapping TDP-43, AD and PD pathologies start in childhood in urbanites with high exposures to PM2.5 and UFPM. Early, sustained exposures to PM air pollution represent a high risk for developing brains and MMC UFPM emissions sources ought to be clearly identified, regulated, monitored and controlled. Prevention of deadly neurologic diseases associated with air pollution ought to be a public health priority and preventive medicine is key.

Linking the phenotype of SNCA Triplication with PET-MRI imaging pattern and alpha-synuclein CSF seeding.

Lewy-body pathology with aggregation of abnormal conformations of the protein alpha-synuclein (α-Syn) represent the histopathological hallmarks of Parkinson's disease (PD). Genetic prototypes such as PD due to mutations in the alpha-synuclein gene (SNCA) offer the opportunity to evaluate α-Syn-related profiles in patient-derived biomaterial. We identified a family with a SNCA triplication and assessed the index patient for CSF α-Syn seeding capacity and levels of total α-Syn along with other neurodegenerative CSF markers (Aß1-42, total-Tau, phospho-Tau, NFL). As no published CSF data in patients with SNCA triplication are available, we descriptively compared his CSF profiles to those of sporadic PD patients and PD patients with GBA mutations as these are also specifically associated with prominent α-Syn pathology. Additionally, skin biopsies with staining for phospho-α-Syn were done. To assess cerebral glucose metabolism and brain atrophy combined positron emission tomography and magnetic resonance imaging ([18F]FDG-PET/MRI) was performed. Age at onset was 24 years and motor impairment was accompanied by prominent non-motor symptoms with early development of dementia, depression, REM sleep behavior disorder, hyposmia, and dysautonomia. Correspondingly, PET-MRI showed hypometabolism and atrophy in frontal, temporoparietal and occipital regions. CSF levels of total α-Syn were threefold higher and RT-QuIC showed remarkable α-Syn seeding activity in all kinetic categories in the SNCATriplication patient compared to patients with GBA mutations. Our results are consistent with findings that not only mutant forms but also overexpression of the wild-type α-Syn protein lead to PD and PD dementia and show a striking CSF α-Syn seeding profile, thus substantiating the role of RT-QuIC as a specific in vivo biomarker of α-Syn brain pathology.

CSF and Serum Levels of Inflammatory Markers in PD: Sparse Correlation, Sex Differences and Association With Neurodegenerative Biomarkers.

Background: An involvement of the central-nervous and peripheral, innate and adaptive immune system in the pathogenesis of Parkinson's disease (PD) is nowadays well established. Objectives: We face several open questions in preparation of clinical trials aiming at disease-modification by targeting the immune system: Do peripheral (blood) inflammatory profiles reflect central (CSF) inflammatory processes? Are blood/CSF inflammatory markers associated with CSF levels of neurodegenerative/PD-specific biomarkers? Methods: Using a multiplex assay we assessed 41 inflammatory markers in CSF/serum pairs in 453 sporadic PD patients. We analyzed CSF/serum correlation as well as associations of inflammatory markers with clinical outcome measures (UPDRS-III, H&Y, MoCA) and with CSF levels of α-synuclein, Aß1-42, t-Tau, p181-Tau and NFL. All analyses were stratified by sex as the immune system shows relevant sex-specific differences. Results: Correlations between CSF and serum were sparse and detected in only 25% (9 out of 36) of the analysable inflammatory markers in male PD patients and in only 38% (12 out of 32) of female PD patients. The most important pro-inflammatory mediators associated with motor and cognitive decline as well as with neurodegenerative/PD-specific biomarkers were FABP, ICAM-1, IL-8, MCP-1, MIP-1-beta, and SCF. Results were more robust for CSF than for serum. Interpretation: Levels of central-nervous and peripheral inflammatory markers might be regulated independently of each other with CSF inflammatory markers reflecting CNS pathology more accurately than peripheral markers. These findings along with sex-specific characteristics have to be considered when designing clinical trials aiming at disease-modification by targeting the immune system.

Association between CSF alpha-synuclein seeding activity and genetic status in Parkinson's disease and dementia with Lewy bodies.

The clinicopathological heterogeneity in Lewy-body diseases (LBD) highlights the need for pathology-driven biomarkers in-vivo. Misfolded alpha-synuclein (α-Syn) is a lead candidate based on its crucial role in disease pathophysiology. Real-time quaking-induced conversion (RT-QuIC) analysis of CSF has recently shown high sensitivity and specificity for the detection of misfolded α-Syn in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this study we performed the CSF RT-QuIC assay in 236 PD and 49 DLB patients enriched for different genetic forms with mutations in GBA, parkin, PINK1, DJ1, and LRRK2. A subgroup of 100 PD patients was also analysed longitudinally. We correlated kinetic seeding parameters of RT-QuIC with genetic status and CSF protein levels of molecular pathways linked to α-Syn proteostasis. Overall, 85% of PD and 86% of DLB patients showed positive RT-QuIC α-Syn seeding activity. Seeding profiles were significantly associated with mutation status across the spectrum of genetic LBD. In PD patients, we detected positive α-Syn seeding in 93% of patients carrying severe GBA mutations, in 78% with LRRK2 mutations, in 59% carrying heterozygous mutations in recessive genes, and in none of those with bi-allelic mutations in recessive genes. Among PD patients, those with severe GBA mutations showed the highest seeding activity based on RT-QuIC kinetic parameters and the highest proportion of samples with 4 out of 4 positive replicates. In DLB patients, 100% with GBA mutations showed positive α-Syn seeding compared to 79% of wildtype DLB. Moreover, we found an association between α-Syn seeding activity and reduced CSF levels of proteins linked to α-Syn proteostasis, specifically lysosome-associated membrane glycoprotein 2 and neurosecretory protein VGF.These findings highlight the value of α-Syn seeding activity as an in-vivo marker of Lewy-body pathology and support its use for patient stratification in clinical trials targeting α-Syn.

CSF Protein Level of Neurotransmitter Secretion, Synaptic Plasticity, and Autophagy in PD and DLB.

BACKGROUND: Molecular pathways associated with α-synuclein proteostasis have been detected in genetic studies and in cell models and include autophagy, ubiquitin-proteasome system, mitochondrial homeostasis, and synaptic plasticity. However, we lack biomarkers that are representative for these pathways in human biofluids. OBJECTIVE: The objective of this study was to evaluate CSF protein profiles of pathways related to α-synuclein proteostasis. METHODS: We assessed CSF protein profiles associated with neurotransmitter secretion, synapse plasticity, and autophagy in 2 monocentric cohorts with α-synucleinopathy (385 PD patients and 67 DLB patients). We included 80 PD patients and 17 DLB patients with variants in the glucocerebrosidase gene to serve as proxy for accelerated α-synuclein pathology with pronounced clinical trajectories. RESULTS: (1) Proteins associated with neurotransmitter secretion, synaptic plasticity, and endolysosomal autophagy were lower in PD and DLB patients compared with healthy controls. (2) These patterns were more pronounced in DLB than in PD patients, accentuated by GBA variant status in both entities. (3) CSF levels of these proteins were positively associated with CSF levels of total α-synuclein, with lower levels of proteostasis proteins related to lower levels of total α-synuclein. (4) These findings could be confirmed longitudinally. PD patients with low CSF profiles of proteostasis proteins showed lower CSF levels of α-synuclein longitudinally compared with PD patients with a normal proteostasis profile. CONCLUSION: CSF proteins associated with neurotransmitter secretion, synaptic plasticity, and endolysosomal autophagy might serve as biomarkers related to α-synuclein proteostasis in PD and DLB. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Mutation Matters: CSF Profiles of GCase, Sphingolipids, α-Synuclein in PDGBA.

BACKGROUND: With pathway-specific trials in PD associated with variants in the glucocerebrosidase gene (PDGBA ) under way, we need markers that confirm the impact of genetic variants in patient-derived biofluids in order to allow patient stratification merely based on genetics and that might serve as biochemical read-out for target engagement. OBJECTIVE: To explore GBA-pathway-specific biomarker profiles cross-sectionally (TUEPAC-MIGAP, PPMI) and longitudinally (PPMI). METHODS: We measured enzyme activity of the lysosomal glucocerebrosidase, CSF levels of glucosylceramides (upstream substrate of glucocerebrosidase), CSF levels of ceramides (downstream product of glucocerebrosidase), lactosylceramides, sphingosines, sphingomyelin (by-products) and CSF levels of total α-synuclein in PDGBA patients compared to PDGBA_wildtype patients. RESULTS: Cross-sectionally in both cohorts and longitudinally in PPMI: (1) glucocerebrosidase activity was significantly lower in PDGBA compared to PDGBA_wildtype . (2) CSF levels of upstream substrates (glucosylceramides species) were higher in PDGBA compared to PDGBA_wildtype . (3) CSF levels of total α-synuclein were lower in PDGBA compared to PDGBA_wildtype . All of these findings were most pronounced in PDGBA with severe mutations (PDGBA_severe ). Cross-sectionally in TUEPAC-MIGAP and longitudinally in PPMI, CSF levels of downstream-products (ceramides) were higher in PDGBA_severe . Cross-sectionally in TUEPAC-MIGAP by-products sphinganine and sphingosine-1-phosphate and longitudinally in PPMI species of by-products lactosylceramides and sphingomyelin were higher in PDGBA_severe . INTERPRETATION: These findings confirm that GBA mutations have a relevant functional impact on biomarker profiles in patients. Bridging the gap between genetics and biochemical profiles now allows patient stratification for clinical trials merely based on mutation status. Importantly, all findings were most prominent in PDGBA with severe variants. © 2021 International Parkinson and Movement Disorder Society.

Parkinson's Disease: Glucocerebrosidase 1 Mutation Severity Is Associated with CSF Alpha-Synuclein Profiles.

BACKGROUND: Mutations in the gene glucocerebrosidase (GBA1) are specifically associated with alpha-synucleinopathies, namely, Parkinson's disease (PD) and dementia with Lewy bodies. As disease-modifying treatment options such as alpha-synuclein lowering compounds are under way, patient stratification according to alpha-synuclein-specific enrichment strategies, possibly reflected by cerebrospinal fluid (CSF) profiles, is a much needed prerequisite. OBJECTIVE: Are GBA1 mutations associated with a CSF alpha-synuclein profile in PD? METHODS: Screening of the GBA1 gene and analysis of CSF levels of total alpha-synuclein were performed in 80 PDGBA , 80 PDGBA _wildtype and 39 healthy controls cross-sectionally. Subgroup analyses based on mutation severity was done for PDGBA . RESULTS: Patients carrying severe GBA1 mutations showed (1) an earlier age at onset, (2) more pronounced cognitive decline and higher prevalence of rapid eye movement sleep behavior disorder, and (3) reduced CSF levels of total alpha-synuclein. CONCLUSION: The effects of GBA1 mutations on CSF alpha-synuclein profiles and phenotypical characteristics seem dependent on GBA1 mutation severity. © 2019 International Parkinson and Movement Disorder Society.

Cerebrospinal fluid non-phosphorylated tau in the differential diagnosis of Creutzfeldt-Jakob disease: a comparative prospective study with 14-3-3.

Cerebrospinal fluid (CSF) non-phosphorylated tau (non-p-tau) is increased in sporadic Creutzfeldt-Jakob disease (CJD), but its accuracy in the differential diagnosis has not been previously established. Here, we first used a retrospective cohort of non-CJD (n = 135) and CJD (n = 137) cases to determine the optimal cutoff point for the discrimination of CJD cases. Next, we prospectively quantified non-p-tau and 14-3-3 protein in a cohort of 1427 cases received for CSF testing at the German National Reference Center for transmissible spongiform encephalopathies. Among them, 36 were subsequently diagnosed as CJD. The diagnostic accuracy of both proteins discriminating CJD cases was evaluated. Using a cutoff of 650 pg/mL, non-p-tau displayed 94.39% accuracy in discriminating CJD cases, while 92.92% accuracy was achieved by 14-3-3 using a cutoff of 20,000 AU/mL. Diagnostic test evaluation for both proteins showed a slightly better performance of non-p-tau compared to 14-3-3. The two biomarkers' concentrations showed a significant positive correlation, both in the total population and in CJD cases (p < 0.001). Finally, the analysis of CSF non-p-tau concentrations when undergoing pre-analytical factors showed high stability in front of temperature storage and freeze/thaw cycles. Therefore, we conclude that when used in the appropriate clinical context of a prion disease surveillance center, non-p-tau is a highly sensitive and specific diagnostic marker for CJD.

Dementia with lewy bodies: GBA1 mutations are associated with cerebrospinal fluid alpha-synuclein profile.

BACKGROUND: Patients with dementia with Lewy bodies reveal a variable pathology including alpha-synuclein, amyloid-beta, and Tau. Mutations in GBA1 are specifically associated with synucleinopathies. PD patients with GBA1 mutations show reduced CSF levels of total alpha-synuclein. OBJECTIVE: Whether GBA1 mutations are associated with a CSF alpha-synuclein profile in dementia with Lewy bodies. METHODS: Screening of the GBA1 gene and single-nucleotide polymorphisms in SNCA rs356220, APOE rs429358, and MAPT rs1052587 as well as CSF levels of total alpha-synuclein, amyloid-beta1-42 , total-Tau, phospho-Tau, and neurofilament light chain were assessed in 100 dementia with Lewy bodies and 39 controls cross-sectionally. RESULTS: Severity of GBA1 mutations was associated with a younger age at onset and higher prevalence of rapid eye movement sleep behavior disorder. CSF levels of total alpha-synuclein were lowest in DLBGBA_pathogenic compared to DLBGBA_mild and DLBGBA_wildtype . CONCLUSION: Similar to PD, pathogenic GBA1 mutations seem to be associated with CSF alpha-synuclein profiles in dementia with Lewy bodies. That might be useful for patient stratification for specific alpha-synuclein-lowering compounds. © 2019 International Parkinson and Movement Disorder Society.

Validation of a prototype tau Thr231 phosphorylation CSF ELISA as a potential biomarker for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques (senile plaques) and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein. This process leads to neuronal degradation and neuronal death. Phosphorylation of tau protein at threonine 231 (p-tau231) has been shown to be characteristic in post-mortem brain tissue of patients with AD and it can be sensitively detected in cerebrospinal fluid (CSF). Therefore, it may serve as a biomarker to support the diagnosis of AD. In this study, we analysed how well p-tau231 could differentiate between patients suffering from dementia either due or not due to AD by a sandwich enzyme immunoassay. CSF p-tau231 was significantly higher in patients with dementia due to AD than in those with dementia due to other causes. In addition, we studied different factors affecting p-tau231 levels in CSF. We found that apolipoprotein E genotype influences p-tau231 CSF levels. Gender and age did not affect p-tau231 levels in CSF. Our findings indicate that p-tau231 levels in CSF can be a valuable marker for the clinical diagnosis of AD.

Non-Phosphorylated Tau in Cerebrospinal Fluid is a Marker of Alzheimer's Disease Continuum in Young Urbanites Exposed to Air Pollution.

Long-term exposure to fine particulate matter (PM2.5) and ozone (O3) above USEPA standards is associated with Alzheimer's disease (AD) risk. Metropolitan Mexico City (MMC) children exhibit subcortical pretangles in infancy and cortical tau pre-tangles, NFTs, and amyloid phases 1-2 by the 2nd decade. Given their AD continuum, we measured in 507 normal cerebrospinal fluid (CSF) samples (MMC 354, controls 153, 12.82±6.73 y), a high affinity monoclonal non-phosphorylated tau antibody (non-P-Tau), as a potential biomarker of AD and axonal damage. In 81 samples, we also measured total tau (T-Tau), tau phosphorylated at threonine 181 (P-Tau), amyloid-ß1-42, BDNF, and vitamin D. We documented by electron microscopy myelinated axonal size and the pathology associated with combustion-derived nanoparticles (CDNPs) in anterior cingulate cortex white matter in 6 young residents (16.25±3.34 y). Non-P-Tau showed a strong increase with age significantly faster among MMC versus controls (p = 0.0055). Aß1 - 42 and BDNF concentrations were lower in MMC children (p = 0.002 and 0.03, respectively). Anterior cingulate cortex showed a significant decrease (p = <0.0001) in the average axonal size and CDNPs were associated with organelle pathology. Significant age increases in non-P-Tau support tau changes early in a population with axonal pathology and evolving AD hallmarks in the first two decades of life. Non-P-Tau is an early biomarker of axonal damage and potentially valuable to monitor progressive longitudinal changes along with AD multianalyte classical CSF markers. Neuroprotection of young urbanites with PM2.5 and CDNPs exposures ought to be a public health priority to halt the development of AD in the first two decades of life.