CSF assays to detect patients with seeding- competent aggregates of Abeta, tau and alpha- synuclein Claudio Soto, PhD Mitchell Center for Alzheimer’s disease and Related Brain Disorders, Dept of Neurology McGovern Medical School, University of Texas at Houston and Amprion, Inc
Misfolded Aggregates deposited in the brain Alzheimer’s disease Parkinson’s disease Protein Misfolding and aggregation Prion diseases Huntington’s disease Soto (2003) Nature Amyothropic lateral sclerosis Rev Neurosci. 4:49-60
Protein misfolding in Neurodegenerative diseases Amyloid fibrils Protofibrils Misfolded Soluble oligomers protein Amyloid plaques Tissue damage Cellular dysfunction
Detection of oligomers: Opportunities and challenges Opportunities Formation of misfolded protein oligomers is possibly the earliest pathological event in neurodegenerative diseases and likely begins decades before clinical symptoms. Misfolded oligomers are thought to be the most biologically active structures in neurodegeneration. Soluble oligomers are likely circulating in biological fluids, offering an opportunity for non-invasive detection. Challenges Misfolded oligomers are highly heterogeneous in size, structure and biological activity. Some oligomers might be on-pathway and others off-pathway in the amyloid fibrillization process. Misfolded oligomers are likely transient, unstable and exist in a much lower concentration than the respective normal monomeric proteins.
How to detect small quantities of misfolded proteins in biological fluids of patients affected by neurodegenerative diseases? Our strategy is to use the ability of misfolded protein aggregates to seed the conversion of the normal protein to enable their high sensitive and specific detection in biological fluids.
Our strategy for sensitive detection Our strategy is to use the ability of misfolded oligomers to seed polymerization of monomeric protein to enable their high sensitivity detection. No seeds + patient’s samples Aggregation Patient’s samples containing seeds No seeds Time
Protein Misfolding Cyclic Amplification (PMCA) Seeds + Incubation Fragmentation Incubation Fragmentation Growing Growing Multiplication Multiplication of units of units of units of units Normal protein Soto et al. (2002) Trends Neurosci. 25:390-394
Applications of PMCA For Prion diseases
Application of PMCA for sensitive detection of prions
Applications of PMCA for Detection of Amyloid-beta Oligomers in Alzheimer’s disease
Alzheimer’s disease neurological alterations Brain atrophy Macroscopic changes Microscopic changes
Preparation of Synthetic A b Oligomers 0h 5h 10h 24h 200 nm Time (h) 0 5 170Kda 4KDa Salvadores et al. (2014) Cell Reports 7: 261
Current status of A β -PMCA Limit of detection below 10 atto-moles
Detection of A b Oligomers by A β -PMCA in CSF Alzheimer’s disease (AD) Non-Neurodegenerative Controls (NND) Non-AD Neurodegenerative Controls (NAND) *** *** *** *** Salvadores et al. (2014) Cell Reports 7: 261
Sensitivity and Specificity in CSF samples Estimation of sensitivity, specificity and predictive value for A β -PMCA using CSF samples Positive Negative Sensitivity 2 Specificity 2 Groups Predictive Predictive Value 2 Value 2 AD vs NAND 100.0% 94.6% 96.2% 100.0% AD vs NND 90.0% 84.2% 88.2% 86.5% AD vs All 3 90.0% 92.0% 88.2% 93.2% AD n=50 NND n=37 NAND n=41 (7 PD, 5 ALS, 6 FTD, 5 PSP, 4 HD, 4 DLB, 5 SCA, 5 PPA) Salvadores et al. (2014) Cell Reports 7: 261
Specificity against samples that may contain other seeds *** *** FTD: Frontotemporal dementia (Tau aggregates) PD: Parkinson disease and Lewy bodies dementia ( α -synuclein aggregates) HD: Huntington’s disease (Huntingtin aggregates) ALS: Amyotrophic lateral sclerosis (SOD and TDP43 aggregates)
Specificity of A β -PMCA assay against cross-seeding Studies of A β -PMCA specificity using synthetic aggregates implicated in the two most prevalent protein misfolding diseases besides AD, i.e. Parkinson disease and type 2 diabetes associated to the aggregation of α -synuclein and amylin, respectively.
Towards a blood-based diagnosis of AD Blood represents the most convenient fluid for a biochemical diagnosis of Alzheimer’s disease Why? ✓ Blood offers the best option for a routine, non-invasive test ✓ It is very well accepted that infectious prions are present in blood of animals and humans and can be detected by PMCA ✓ A β has been shown to be present in blood and contribute to brain pathology ✓ A β can cross the blood-brain barrier in both directions ✓ Labeled A β injected in blood can be retrieved in brain plaques But.. It is technically very challenging ✓ Blood is a very complex fluid with many other component that interfere with A β aggregation assay ✓ It is likely that the amount of misfolded A β oligomers circulating in blood is very low and its detection will be confounded by the larger concentration of soluble A β ✓ Misfolded A β oligomers are presumably bound to other proteins, making difficult its detection
Plasma A β -PMCA requires a pre-capture step Strategy 1: Using antibody-coated plates 100 µl/well in duplicates Pre clearing the Blood Plasma A β -PMCA 3000 rpm X 15 min 200 µl BP 1:1 dilution in ELISA plates coated PBS T(0.1%) + PI with sequence or conformational antibodies Strategy 2: Immuno-precipitation and concentration by antibody-coated beads Incubation with antibody Pre-clearing the (sequence or conformational) 1:1 dilution Blood Plasma coated beads 16 h at 22 °C in 2X PBS + PI + 1% NP40 500 µl BP Beads washed re-suspended in 20 ul of aggregation buffer 10 ul added Into two wells A β -PMCA
Sensitivity of A β -PMCA in spiked plasma Aggregation, % Time, h Minimum detectable amount of A β oligomers = 20pg, equivalent to 1.1 x 10 -16 moles (assuming an average molecular weight of 170KDa for the oligomers) and extent of seeding is proportional to the quantity of seeds
Detection of A β oligomers in AD plasma Alzheimer’s disease patients *** 93.3% sensitivity; 90% specificity
Applications of PMCA for Detection of Tau Oligomers
Cyclic Amplification of Tau Misfolding (Tau-PMCA) T 50 , hours Initial detection limit 0.125 pg of Tau seeds, which is equivalent to 1 atto- mol (assuming a MW of 135K). Direct relationship between the amount of oligomers and the parameters of Tau-PMCA
Specificity of Tau-PMCA assay against cross-seeding
Reproducibility of the Tau-PMCA assay Experiments were done in triplicate with 2 different Tau seeds, at 4 distinct times, in buffer or CSF, with or without freezing/thawing, and with 5 different concentrations of seeds. No significant differences were observed in any condition.
Preliminary results with human CSF samples ** ** 3 0 0 0 , n t i o i t s a 2 0 0 0 n g u r e e g c n g a e c m s r e u i m o 1 0 0 0 f l u x a M 0 (4 PSP, 1 FTD, 5 CBD and 1 CTE)
Applications of PMCA for Detection of α -Synuclein Oligomers
Brain alterations in Parkinson’s disease α -synuclein aggregates in Lewy bodies
Preparation of Synthetic α -syn aggregates KDa 135 Large 120 oligomers 75 22 Monomer 17 240 h 96 h O h 50 nm 50 nm 50 nm Shahnawaz et al. (2017) JAMA Neurol 74: 163
α Syn-PMCA in automatic machine Detection limit below 2 pg (15 atto-mol, assuming a MW of 135K). Direct relationship between the amount of oligomers and the parameters of α Syn-PMCA. This system allow continuous monitoring of the entire plate and using a plate stacker we can run up to 20 plates per machine.
Specificity of α Syn-PMCA No signal was detectable when the reaction was incubated with A β or Tau oligomers. The concentration of seeds added was very high (higher than the highest amount shown in the previous graph). These seeds produce a very large signal in the respective A β -PMCA and Tau-PMCA assays. The results indicate that α Syn-PMCA is very specific to detect α Syn oligomers. Shahnawaz et al. (2017) JAMA Neurol 74: 163
α Syn-PMCA in CSF Aggregation, % Aggregation, % Time, h Time, h Using optimized conditions, we can detect as little as 0.02 pg of α Syn oligomers in CSF, which translate to around 0.15 atto-mols (assuming a MW of 135 KDa). Clear signal was observed in the samples from patients affected by Parkinson’s disease and no signal in controls. α Syn-PMCA signal can be reduced by immuno-depletion of oligomers. Shahnawaz et al. (2017) JAMA Neurol 74: 163
Results of a blinded study in CSF Parkinson’s Disease Fluorescence Disease Controls *** # # # # 86% sensitivity # These two patients developed symptoms of PD 1 and 4 years after sample was collected, one of them was confirmed by autopsy. Shahnawaz et al. (2017) JAMA Neurol 74: 163
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