Evaluation of plasma levels of NFL, GFAP, UCHL1 and tau as Parkinson's disease biomarkers using multiplexed single molecule counting.

Objective biomarkers for Parkinson's Disease (PD) could aid early and specific diagnosis, effective monitoring of disease progression, and improved design and interpretation of clinical trials. Although alpha-synuclein remains a biomarker candidate of interest, the multifactorial and heterogenous nature of PD highlights the need for a PD biomarker panel. Ideal biomarker candidates include markers that are detectable in easily accessible samples, (ideally blood) and that reflect the underlying pathological process of PD. In the present study, we explored the diagnostic and prognostic PD biomarker potential of the SIMOA neurology 4-plex-A biomarker panel, which included neurofilament light (NFL), glial fibrillary acid protein (GFAP), tau and ubiquitin C-terminal hydrolase L1 (UCHL-1). We initially performed a serum vs plasma comparative study to determine the most suitable blood-based matrix for the measurement of these proteins in a multiplexed assay. The levels of NFL and GFAP in plasma and serum were highly correlated (Spearman rho-0.923, p < 0.0001 and rho = 0.825, p < 0.001 respectively). In contrast, the levels of tau were significantly higher in plasma compared to serum samples (p < 0.0001) with no correlation between sample type (Spearman p > 0.05). The neurology 4-plex-A panel, along with plasma alpha-synuclein was then assessed in a cross-sectional cohort of 29 PD patients and 30 controls. Plasma NFL levels positively correlated with both GFAP and alpha-synuclein levels (rho = 0.721, p < 0.0001 and rho = 0.390, p < 0.05 respectively). As diagnostic biomarkers, the control and PD groups did not differ in their mean NFL, GFAP, tau or UCHL-1 plasma levels (t test p > 0.05). As disease state biomarkers, motor severity (MDS-UPDRS III) correlated with increased NFL (rho = 0.646, p < 0.0001), GFAP (rho = 0.450, p < 0.05) and alpha-synuclein levels (rho = 0.406, p < 0.05), while motor stage (Hoehn and Yahr) correlated with increased NFL (rho = 0.455, p < 0.05) and GFAP (rho = 0.549, p < 0.01) but not alpha-synuclein levels (p > 0.05). In conclusion, plasma was determined to be most suitable blood-based matrix for multiplexing the neurology 4-plex-A panel. Given their correlation with motor features of PD, NFL and GFAP appear to be promising disease state biomarker candidates and further longitudinal validation of these two proteins as blood-based biomarkers for PD progression is warranted.

Comparison of Different Platform Immunoassays for the Measurement of Plasma Alpha-Synuclein in Parkinson's Disease Patients.

BACKGROUND: The identification of reliable biomarkers in Parkinson's disease (PD) would provide much needed diagnostic accuracy, a means of monitoring progression, objectively measuring treatment response, and potentially allowing patient stratification within clinical trials. Whilst the assessment of total alpha-synuclein in biofluids has been identified as a promising biomarker, conflicting trends in these levels across patient plasma samples relative to controls has limited its use. Different commercially available assay platforms that have been used to measure alpha-synuclein may contribute to different study outcomes. OBJECTIVE: To compare different platform immunoassays for the measurement of total alpha-synuclein using the same plasma samples from 49 PD patients and 47 controls. METHODS: Total plasma alpha-synuclein concentrations were assessed using the BioLegend, MesoScale Discovery, and Quanterix platform in plasma samples from PD patients and matched controls. RESULTS: A significant increase in total plasma alpha-synuclein was observed in PD patients using the Biolegend (10%), Mesoscale Discovery (13%) and Quanterix (39%) assays. The Mesoscale Discovery and Quanterix assays showed the strongest correlations (r = 0.78, p < 0.0001) with each other, whilst the Quanterix platform demonstrated the lowest variation and highest effect size. Inclusion of age, sex and hemoglobin levels as covariates in the analysis of total alpha-synuclein improved the ability of all three immunoassays to detect a significant difference between patients and controls. CONCLUSION: All three immunoassays were sensitive enough to detect group level differences between PD patients and controls, with the largest effect size observed with the Quanterix assay. These results may help inform assay choices in ongoing clinical trials.

Evidence supporting oxidative stress in a moderately affected area of the brain in Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) remains to be elucidated. Oxidative damage and excessive beta-amyloid oligomers are components of disease progression but it is unclear how these factors are temporally related. At post mortem, the superior temporal gyrus (STG) of AD cases contains plaques, but displays few tangles and only moderate neuronal loss. The STG at post mortem may represent a brain region that is in the early stages of AD or alternately a region resistant to AD pathogenesis. We evaluated expression profiles and activity of endogenous anti-oxidants, oxidative damage and caspase activity in the STG of apolipoprotein ε4-matched human AD cases and controls. Total superoxide dismutase (SOD) activity was increased, whereas total glutathione peroxidase (GPX), catalase (CAT) and peroxiredoxin (Prx) activities, were decreased in the AD-STG, suggesting that hydrogen peroxide accumulates in this brain region. Transcripts of the transcription factor NFE2L2 and inducible HMOX1, were also increased in the AD-STG, and this corresponded to increased Nuclear factor erythroid 2-related factor (NRF-2) and total heme-oxygenase (HO) activity. The protein oxidation marker 4-hydroxynonenal (4-HNE), remained unchanged in the AD-STG. Similarly, caspase activity was unaltered, suggesting that subtle redox imbalances in early to moderate stages of AD do not impact STG viability.

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

Oxidative stress in Alzheimer's disease: Primary villain or physiological by-product?

The prevalence of Alzheimer's disease (AD) is increasing rapidly worldwide due to an ageing population and largely ineffective treatments. In AD cognitive decline is due to progressive neuron loss that begins in the medial temporal lobe and spreads through many brain regions. Despite intense research the pathogenesis of the common sporadic form of AD remains largely unknown. The popular amyloid cascade hypothesis suggests that the accumulation of soluble oligomers of beta amyloid peptides (Aß) initiates a series of events that cause neuronal loss. Among their putative toxic effects, Aß oligomers are thought to act as pro-oxidants combining with redox-active metals to produce excessive reactive oxygen and nitrogen species. However, to date the experimental therapies that reduce Aß load in AD have failed to halt cognitive decline. Another hypothesis proposed by the late Mark Smith and colleagues is that oxidative stress, rather than Aß, precipitates the pathogenesis of AD. That is, Aß and microtubule-associated protein tau are upregulated to address the redox imbalance in the AD brain. As the disease progresses, excess Aß and tau oligomerise to further accelerate the disease process. Here, we discuss redox balance in the human brain and how this balance is affected by ageing. We then discuss where oxidative stress is most likely to act in the disease process and the potential for intervention to reduce its effects.