TDP-43 seeding activity in the olfactory mucosa of patients with amyotrophic lateral sclerosis

Abstract

Background: In recent years, the seed amplification assay (SAA) has enabled the identification of pathological TDP-43 in the cerebrospinal fluid (CSF) and olfactory mucosa (OM) of patients with genetic forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we investigated the seeding activity of TDP-43 in OM samples collected from patients with sporadic ALS.

Methods: OM samples were collected from patients with (a) sporadic motor neuron diseases (MND), including spinal ALS (n = 35), bulbar ALS (n = 18), primary lateral sclerosis (n = 10), and facial onset sensory and motor neuronopathy (n = 2); (b) genetic MND, including carriers of C9orf72^exp (n = 6), TARDBP (n = 4), SQSTM1 (n = 3), C9orf72^exp + SQSTM1 (n = 1), OPTN (n = 1), GLE1 (n = 1), FUS (n = 1) and SOD1 (n = 4) mutations; (c) other neurodegenerative disorders (OND), including Alzheimer's disease (n = 3), dementia with Lewy bodies (n = 8) and multiple system atrophy (n = 6); and (d) control subjects (n = 22). All samples were subjected to SAA analysis for TDP-43 (TDP-43_SAA). Plasmatic levels of TDP-43 and neurofilament-light chain (NfL) were also assessed in a selected number of patients.

Results: TDP-43_SAA was positive in 29/65 patients with sporadic MND, 9/21 patients with genetic MND, 6/17 OND patients and 3/22 controls. Surprisingly, one presymptomatic individual also tested positive. As expected, OM of genetic non-TDP-43-related MND tested negative. Interestingly, fluorescence values from non-MND samples that tested positive were consistently and significantly lower than those obtained with sporadic and genetic MND. Furthermore, among TDP-43-positive samples, the lag phase observed in MND patients was significantly longer than that in non-MND patients. Plasma TDP-43 levels were significantly higher in sporadic MND patients compared to controls and decreased as the disease progressed. Similarly, plasma NfL levels were higher in both sporadic and genetic MND patients and positively correlated with disease progression rate (ΔFS). No significant correlations were detected between TDP-43_SAA findings and the biological, clinical, or neuropsychological parameters considered.

Conclusions: The OM of a subset of patients with sporadic MND can trigger seeding activity for TDP-43, as previously observed in genetic MND. Thus, TDP-43_SAA analysis of OM can improve the clinical characterization of ALS across different phenotypes and enhance our understanding of these diseases. Finally, plasma TDP-43 could serve as a potential biomarker for monitoring disease progression. However, further research is needed to confirm and expand these findings.

Keywords: Amyotrophic lateral sclerosis; Neurodegeneration; Olfactory mucosa; Peripheral biomarkers; Seed amplification assay; TDP-43.

Fig. 1

TDP-43_SAA results for OM samples from sporadic MND cases. TDP-43_SAA showed seeding activity in 43% of the sALS cases, 39% of the bALS cases, 60% of the PLS cases, 50% of the FOSMN cases, 50% of the DLB cases, and 67% of the AD cases. No seeding activity was observed in the MSA samples, whereas 14% of the CTRL samples tested positive. Abbreviations: sALS = spinal-onset amyotrophic lateral sclerosis; bALS = bulbar-onset amyotrophic lateral sclerosis; PLS = primary lateral sclerosis; FOSMN = facial onset sensory and motor neuronopathy; MSA = multiple system atrophy; DLB = dementia with Lewy body; AD = Alzheimer's disease; CTRL = controls; AU = arbitrary units; h = hours

Fig. 2

TDP-43_SAA results for OM samples from genetic MND cases. TDP-43_SAA showed seeding activity in 50% of TARDBP cases (green curves), 67% of SQSTM1 cases (light blue curves), 100% of OPTN (pink curve) and GLE1 cases (orange curve) and 50% of C9orf72^exp cases (dark blue curves) (including a presymptomatic case, dotted dark blue curve). Gray lines indicate cases in which no seeding was observed. Abbreviations: MND = motor neuron disease; C9orf72 = chromosome 9 open reading frame 72; SQSTM1 = sequestosome 1; SOD1 = superoxide dismutase type 1; TARDBP = TAR DNA-binding protein; GLE1 = GLE1 RNA export mediator; OPTN = optineurin; AU = arbitrary units; h = hours

Fig. 3

Boxplots showing the mean last fluorescence levels (A) and lag phases (B) of OM samples that tested positive for TDP-43_SAA. The average last fluorescence value observed in patients who tested positive for TDP-43_SAA (including OND patients and CTRLs, referred to as non-MND patients) was significantly greater in MND patients than in non-MND patients (A). Similarly, the average lag phase of MND patients was significantly longer than that of non-MND patients (B)

Fig. 4

αSyn_SAA results for OM samples from sporadic MND cases. αSyn_SAA showed seeding activity in 9% of sALS cases and 11% of bALS cases, 67% of MSA cases, 100% of DLB cases and 33% of AD cases. No seeding activity was observed in the PLS, FOSMN or CTRL samples. Abbreviations: sALS = spinal-onset amyotrophic lateral sclerosis; bALS = bulbar-onset amyotrophic lateral sclerosis; PLS = primary lateral sclerosis; FOSMN = facial onset sensory and motor neuronopathy; MSA = multiple system atrophy; DLB = dementia with Lewy body; AD = Alzheimer's disease; CTRL = controls; AU = arbitrary units; h = hours

Fig. 5

αSyn_SAA results for OM samples from genetic MND cases. αSyn_SAA showed seeding activity in 1 C9orf72^exp patient (dark blue curve), 2 SQSTM1 patients (light blue curves) and 1 TARDBP patient (green curve). No seeding activity was detected in C9orf72^exp + SQSTM1, OPTN, GLE1, FUS or SOD1 cases (gray lines). Abbreviations: MND = motor neuron disease; C9orf72^exp = chromosome 9 open reading frame 72 expansion; SQSTM1 = sequestosome 1; SOD1 = superoxide dismutase type 1; TARDBP = TAR DNA-binding protein; AU = arbitrary units; h = hours

Fig. 6

Plasma TDP-43 concentration, plasma NfL concentration, and correlation with clinical variables. Plasma TDP-43 levels in patients with sporadic MND were significantly higher compared to the CTRL group (Wilcoxon rank-sum test, p = 0.0254) (A). eGFR values were within the normal range for all patients with MND, and no statistically significant correlation was observed between eGFR values and TDP-43 levels (r = − 0.173, p = 0.207) (B). TDP-43 levels in MND patients significantly decreased with increasing disease burden, as assessed by the King's Clinical Staging System (ANOVA test, p = 0.011) (C). NfL values in patients with sporadic and genetic MND were significantly elevated compared to the CTRL group (Wilcoxon rank-sum test, both p < 0.0001) (D). No statistically significant correlation was observed between eGFR values and NfL levels (r = 0.018, p = 0.895) (E). NfL levels in MND patients significantly increased with disease progression rate (ΔFS) (ANOVA test, p = 0.003) (F)