ALS plasma biomarkers reveal neurofilament and pTau correlate with disease onset and progression

Abstract

Objective: We performed a pilot screen to assess the utility of the NULISA™ (Nucleic-acid-Linked Immuno-Sandwich Assay) platform in the identification of amyotrophic lateral sclerosis (ALS) biomarkers.

Methods: Plasma from 86 individuals (48 ALS, 18 asymptomatic C9orf72 repeat expansion carriers (AsymC9), and 20 healthy controls) was analyzed via a multiplexed NULISA™ assay that includes 120 neurodegeneration-associated proteins. Statistical analysis of NULISA™ results was performed to identify proteins differentially expressed in plasma and their correlation with disease-associated parameters.

Results: ALS plasma showed elevation of the established biomarkers, neurofilament light chain (NEFL) and neurofilament heavy chain (NEFH). Compared to controls and AsymC9, microtubule-associated protein tau (MAPT), phosphorylated tau 181 (pTau181), phosphorylated tau 217 (pTau217), phosphorylated tau 231 (pTau231), and phosphorylated TDP-43 (pTDP-43) were elevated in ALS. NEFL levels positively correlated with pTau181, pTau217, pTau231, and pTDP-43. MAPT and pTDP-43 were also correlated with pTau181, pTau217 and pTau231. Elevated pTau was negatively correlated with survival and ALSFRS-R. Spinal onset ALS was associated with higher pTau181, pTau217, and pTau231.

Interpretation: We confirm previous reports showing elevated pTau181 in ALS plasma and show elevation of other phosphorylated tau forms, pTau217 and pTau231, typically observed in Alzheimer's disease. We provide preliminary data showing the detection and elevation of pTDP-43-409/410 in a subset of ALS samples compared to healthy controls. Neurofilament and tau levels are highly correlated suggesting their elevation may reflect a common pathology and disease state. Total and phosphorylated tau are correlated with multiple disease measures, such as ALS duration, ALSFRS-R, and site of onset.

Figure 1

Elevation of phosphorylated Tau and TDP‐43 in ALS plasma. NULISA Protein Quantification (NPQ) for each protein is shown on the y‐axis and the limit of detection (LOD) is shown with a dotted line. NPQ for Control, asymptomatic C9, and ALS are compared for (A) NEFL (ANOVA p = 2.0e⁻²⁵), (B) NEFH (ANOVA p = 2.9e⁻²⁵), (C) MAPT (ANOVA p = 0.01), (D) pTau181 (ANOVA p = 1.9e⁻⁹), (E) pTau217 (ANOVA p = 4.3e⁻⁹), (F) pTau231 (ANOVA p = 6.1e⁻¹⁰), (G) TARDP (ANOVA p = 0.29, n.s.) and (H) pTDP‐43 (ANOVA p = 0.036). ROC Curve analysis comparing ALS to healthy controls. (I) NEFL AUC = 0.9979, 95% confidence interval 0.9923–1.000, (J) NEFH AUC = 0.9563, 95% confidence interval [0.9128–0.9997], (K) pTDP‐43 AUC = 0.7810, 95% confidence interval [0.6643–0.8977], (L) pTau181 AUC = 0.9063, 95% confidence interval [0.8357–0.9768, (M) pTau217 AUC = 0.8865, 95% confidence interval [0.8053–0.9676], (N) pTau231 AUC = 0.9125, 95% confidence interval [0.8465–0.9785].

Figure 2

Phosphorylated Tau, Neurofilament and pTDP‐43 are correlated. Scatter plot of NEFL vs phosphorylated tau (A–C) and MAPT vs phosphorylated tau (D–F) and pTDP‐43 vs phosphorylated tau and NEFL (G–J) shown with linear regression including all samples (Controls, Asym C9 and ALS). NEFL is positively correlated with pTau181 (ρ = 0.691, p < 0.0001), pTau217 (ρ = 0.693, p < 0.0001), and pTau231 (ρ = 0.719, p < 0.0001). MAPT is positively correlated with pTau181 (ρ = 0.846, p < 0.0001), pTau217 (ρ = 0.824, p < 0.0001), and pTau231 (ρ = 0.852, p < 0.0001). pTDP‐43 is positively correlated with pTau181 (ρ = 0.397, p < 0.001), pTau217 ρ = 0.419, p < 0.0001), pTau231 (ρ = 0.389, p < 0.001), and NEFL (ρ = 0.291, p < 0.01).

Figure 3

Elevated phosphorylated Tau correlates with disease severity. NEFL, MAPT, pTau181, pTau217, and pTau231 are negatively correlated with ALS disease duration (A–F). ALSFRS‐R was available in 46/48 ALS cases. pTau181, pTau217, pTau231, and NEFL are negatively correlated with ALSFRS‐R (G–J).

Figure 4

Phosphorylated and total tau are higher in spinal onset ALS. Spinal onset (n = 34) and bulbar onset (n = 14) ALS were compared by Student's t‐test. (A–D) pTau181, pTau217, pTau231 and MAPT were significantly higher in spinal onset ALS cases than bulbar onset ALS cases (pTau181 p‐value = 0.003; pTau217 p‐value = 0.016; pTau231 p‐value = 0.002; MAPT p‐value = 0.0025). There was no difference in NEFH and NEFL between spinal and bulbar onset cases (E and F).