Plasma biomarkers of neurodegeneration in patients and high risk subjects with Lewy body disease

Comorbid Alzheimer’s disease (AD) neuropathology is common in Lewy body disease (LBD); however, AD comorbidity in the prodromal phase of LBD remains unclear. This study investigated AD comorbidity in the prodromal and symptomatic phases of LBD by analyzing plasma biomarkers in patients with Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) and individuals at risk of LBD (NaT-PROBE cohort). Patients with PD (PD group, n = 84) and DLB (DLB group, n = 16) and individuals with LBD with ≥ 2 (high-risk group, n = 82) and without (low-risk group, n = 37) prodromal symptoms were enrolled. Plasma amyloid-beta (Aβ) composite was measured using immunoprecipitation-mass spectrometry assays. Plasma phosphorylated tau 181 (p-tau181), neurofilament light chain (NfL), and alpha-synuclein (aSyn) were measured using a single-molecule array. Plasma p-tau181 levels were higher in the PD and DLB groups than in the low-risk group. Aβ composite level was higher in the DLB group than in the high-risk group. AD-related biomarker levels were not elevated in the high-risk group. NfL levels were higher in the high-risk, PD, and DLB groups than in the low-risk group. In the PD group, Aβ composite was associated with cognitive function, p-tau181 with motor function and non-motor symptoms, and NfL with cognitive and motor functions and non-motor symptoms. In the high-risk group, NfL was associated with metaiodobenzylguanidine scintigraphy abnormalities. The PD and DLB groups exhibited comorbid AD neuropathology, though not in the prodromal phase. Elevated plasma NfL levels, even without elevated AD-related plasma biomarker levels, may indicate aSyn-induced neurodegeneration in the LBD prodromal phase.

mild cognitive decline and hyposmia compared with low-risk participants with no prodromal symptoms.Approximately one-third of the high-risk individuals had defects in dopamine transporter (DaT) single-photonemission computed tomography (SPECT) or cardiac metaiodobenzylguanidine (MIBG) scintigraphy, and the prevalence of abnormalities on DaT-SPECT was 4 times higher in the high-risk individuals than that in the lowrisk individuals 3 .
In PD and DLB, limbic and neocortical aSyn pathology is associated with dementia; furthermore, previous postmortem brain studies demonstrated that comorbid Alzheimer's disease (AD) neuropathology is associated with the progression of cognitive impairment.More than 70% of patients with DLB and approximately 50% of patients with PD dementia (PDD) have comorbid AD neuropathology 4,5 .Understanding the temporal progression of comorbid AD neuropathology is crucial for comprehending the motor and cognitive trajectories in LBD, and AD-related molecules may serve as a potential therapeutic target.Recently, AD-related plasma biomarkers, such as amyloid-beta (Aβ) composite (combination biomarker of amyloid-beta precursor protein (APP) 669-711 /Aβ 1-42 and Aβ 1-40 /Aβ 1-42 ratios) and phosphorylated tau 181 (p-tau181), have garnered attention 6,7 .In addition, plasma neurofilament light chain (NfL) is regarded as a reliable biomarker for various neurodegenerative diseases 8 .Although recent studies have examined AD-related plasma biomarkers in patients with PD and DLB 9,10 and plasma NfL in patients with idiopathic RBD 11,12 , there is a lack of detailed studies on AD comorbidity in the prodromal phase of LBD.
This study measured and analyzed four plasma biomarkers, Aβ composite, p-tau181, NfL, and aSyn, in high-risk and low-risk individuals, participating in the NaT-PROBE study, as well as in patients with PD and DLB.

Participant characteristics
There were more male participants in the low-and high-risk groups than in the PD and DLB groups.The PD and DLB group participants were significantly older than the low-and high-risk group participants.Among the high-risk participants, 36.6% had abnormalities in either DaT or MIBG, consistent with the findings in our previous study 3 .All patients with PD and DLB who underwent DaT or MIBG before the study inclusion exhibited these abnormalities.The PD and DLB group participants, though not the high-risk group participants, had worse MoCA-J scores compared with the low-risk group participants.Two patients with PD and three with DLB could not complete the Stroop test, and one patient with DLB could not complete the line orientation test.The average Hoehn and Yahr Scale score was similar between the PD and DLB groups.The PD and DLB group participants, though not those in the high-risk group, had worse MDS-UPDRS III scores.The high-risk participants who were selected based on the SCOPA-AUT, SAOQ, and RBDSQ scores had worse BDI-II, ESS, PDQ-39, and QUIP scores than the low-risk participants.The PD and DLB group participants had worse scores on these questionnaires as well, except for QUIP (Table 1).
The PD-CI group participants were significantly older than the PD-CN group participants.The PD-CI group participants, who were selected based on the MoCA-J scores, had worse line orientation test scores than those of the PD-CN group participants.Compared with the PD-CN group participants, the PD-CI participants had worse RBDSQ, BDI-II, PDQ-39, and QUIP scores, while no significant difference was found in motor function (Supplementary Table 1).

Plasma biomarkers
Pearson's correlation analysis between plasma biomarkers and age exhibited a weak correlation for plasma Aβ composite in the PD group, weak correlations for plasma p-tau181 in the low-risk, high-risk, and PD groups, and moderate correlations for plasma NfL in the high-risk and PD groups (Supplementary Fig. 1).Therefore, all statistical tests regarding plasma biomarkers were adjusted for age.Considering the phenotypic differences of AD between females and males, biomarker values for sex were also adjusted 13 .
Plasma Aβ composite levels in the DLB group were higher than those in the other groups; however, this difference was significant only between the DLB and the high-risk groups (Fig. 1a).Plasma log 10 (p-tau181) levels were significantly higher in the PD and DLB groups than those in the lowand high-risk groups (Fig. 1b).Plasma log 10 (NfL) levels were significantly higher in the high-risk, PD, and DLB groups than those in the low-risk group, with the DLB group exhibiting a pronounced elevation (Fig. 1c).Plasma aSyn/Hb ratios were significantly lower in the PD group than those in the high-risk and DLB groups.Plasma aSyn/Hb ratios were significantly higher in the DLB group than those in the low-risk group (Fig. 1d).Plasma aSyn levels, similar to aSyn/Hb ratios, were significantly lower in the PD group than those in the high-risk and DLB groups, and significantly higher in the DLB group than those in the low-risk group (Supplementary Fig. 2a).Hemoglobin levels were significantly lower in the PD group than those in the high-risk group and significantly lower in the DLB group than those in the low-and high-risk groups (Supplementary Fig. 2b).
The age-adjusted partial correlation analysis to assess the relationships between plasma biomarkers revealed no correlation between the plasma biomarkers in the low-and high-risk groups (Fig. 2a-f).In the PD group, Aβ composite and log 10 (p-tau181) and log 10 (p-tau181) and log 10 (NfL) were weakly correlated (Fig. 2g-i).In the DLB group, Aβ composite and log 10 (p-tau181) were moderately correlated (Fig. 2j-l).
The PD-CI group exhibited a significant increase in plasma Aβ composite and log 10 (p-tau181) levels compared with the PD-CN group (Fig. 3a, b).Although plasma log 10 (NfL) levels tended to be higher in the PD-CI group than in the PD-CN group, the difference was not significant (Fig. 3c).No significant differences were found in the aSyn/Hb ratio between the PD-CI and PD-CN groups (Fig. 3d).

Differences in clinical features and plasma biomarkers of patients with PD and high-risk individuals across AT(N) profiles
Based on the cut-off values for Aβ composite, p-tau181, and NfL, participants were classified as normal/abnormal (−/+) for Aβ (A), tau (T), and neurodegeneration (N).The proportions of A − T − (N)− and A + T + (N)+ were significantly lower and higher, respectively, in the PD and DLB groups than those in the low-risk group.The proportion of A − T − (N)+ in the high-risk group was significantly higher than that in the low-risk group (Table 2).
Patients with PD classified as A+ were older and had a shorter educational history, worse MoCA-J scores, and higher plasma log 10 (p-tau181) levels than those classified as A−.Patients with PD classified as T+ were older and had worse Hoehn and Yahr Scale and MDS-UPDRS III total scores and subscores for bradykinesia and axial signs, and worse SCOPA-AUT, BDI-II, PDQ-39, and QUIP scores than those classified as T−.Patients with PD classified as T+ had higher levels of Aβ composite and log 10 (NfL) than those classified as T−.Patients with PD classified as N+ were older and had worse scores on the MoCA-J and Hoehn and Yahr Scales, worse MDS-UPDRS III total scores and subscores for bradykinesia and axial signs, worse SCOPA-AUT, BDI-II, PDQ-39, and QUIP scores, and higher levels of plasma Aβ composite and log 10 (p-tau181) than those classified as N− (Table 3).
The age-adjusted partial correlation analysis that assessed the relationships between plasma biomarkers and clinical indices (Supplementary Fig. 3) revealed that plasma log 10 (NfL) was weakly correlated with MDS-UPDRS III bradykinesia and axial signs subscores and the SCOPA-AUT and PDQ-39 scores.In addition, plasma log 10 (p-tau181) was weakly correlated with the SCOPA-AUT and PDQ-39 scores, while plasma aSyn/Hb ratio was weakly correlated with the MDS-UPDRS III rigidity subscore.
No significant differences in age, cognitive and motor functions, questionnaire survey scores, or plasma biomarkers were found between the high-risk participants classified as A+ and A−.The high-risk participants classified as T+ were significantly older and had worse scores on the Hoehn and Yahr Scale, MDS-UPDRS III rigidity subscore, and the RBDSQ, and QUIP scales than those classified as T−.The high-risk participants classified as N+ were significantly older and had a higher rate of MIBG abnormalities than those classified as N− (Table 4).The age-adjusted partial correlation analysis revealed no significant correlations between plasma biomarkers and each clinical score (Supplementary Fig. 4).

Discussion
This study measured and analyzed plasma Aβ composite, p-tau181, NfL, and aSyn in patients with PD and DLB and high-and low-risk individuals who were identified in a questionnaire survey on prodromal symptoms of LBD.The results revealed that both PD and DLB groups had increased plasma p-tau181 levels, indicating that comorbid AD neuropathology exists in manifest LBD.In addition, plasma NfL levels were elevated in the highrisk group despite the absence of significant elevation in AD-related plasma biomarker levels such as Aβ composite and p-tau181; thus, plasma NfL levels may reflect aSyn-induced neurodegeneration in the prodromal phase of LBD.
Previous studies demonstrated that higher plasma Aβ composite levels can predict Aβ burden with approximately 90% accuracy when using Pittsburgh compound-B (PIB)-amyloid positron emission tomography (PET) as the standard of truth 6 , and higher plasma p-tau181 levels can predict Aβ and tau positivity on PET 7 .In the present study, although plasma Aβ composite levels were higher in the DLB group than those in the other groups, the difference was only significant between the DLB and the high-risk groups, unlike the increase in plasma p-tau181 levels which was significant in both the PD and DLB groups.Although this incongruity may be a result of the limited statistical power in the multigroup comparison, previous PET studies reported a substantially low incidence of amyloid deposition in PD without dementia 14,15 .Another plasma biomarker study reported that the plasma Aβ 1-42 /Aβ 1-40 ratio was increased in the PD without dementia group compared with that in healthy controls and decreased in the PD with dementia group compared with that in the PD without dementia group 9 .Collectively, these findings suggest that amyloid pathology develops concurrently with cognitive decline in LBD and that p-tau biomarkers are more sensitive than Aβ biomarkers in early PD.
However, in our focused analysis on the PD group, both AD-related plasma biomarker (Aβ composite and p-tau181) levels were significantly higher in the PD-CI group than those in the PD-CN group.This suggests that comorbid AD neuropathology influences the development of cognitive impairment in PD, consistent with previous cerebrospinal fluid (CSF) studies 16 .In the patients with PD, those classified as A+ had worse MoCA-J scores compared with those classified as A−, and those classified as T+ had worse scores on motor function (MDS-UPDRS III subscores on  bradykinesia and axial signs), the SCOPA-AUT, PDQ-39, and QUIP scales compared with those classified as T−.These observations are consistent with a previous CSF study which reported that lower CSF Aβ 1-42 and higher p-tau were associated with delayed memory recall and motor function, respectively 17 .Conversely, the AD-related plasma biomarkers were not elevated in the high-risk group.These results are consistent with those reported in previous studies, namely, that the rate of positive amyloid PET in patients with idiopathic RBD was similar to that in cognitively normal individuals [18][19][20] .Although these findings indicate that AD-related plasma biomarkers become detectable after the manifestation of motor/cognitive symptoms in LBD, comorbid AD neuropathology may subsist at an undetectable level in the prodromal phase and influence disease progression.Therefore, further longitudinal data analysis is required to elucidate the role of AD-related plasma biomarkers on motor function and non-motor symptoms in highrisk individuals.
Elevated plasma NfL level is a reliable biomarker of neurodegeneration in various diseases 8 .Although results of previous cross-sectional studies on PD on the correlation between plasma NfL levels and cognitive and motor functions are inconclusive, those of prospective studies consistently demonstrate a correlation between baseline plasma NfL and worsening cognitive and motor functions 21 .Another study reported that higher baseline NfL levels in patients with idiopathic RBD were associated with worsening cognitive, motor, and autonomic functions and a higher risk of phenoconversion 11 .The present study demonstrated that plasma NfL levels were significantly elevated in the PD, DLB, and high-risk groups compared with those in the low-risk group.In patients with PD, those classified as N+ had worse scores on the MoCA-J, Hoehn and Yahr, MDS-UPDRS III, SCOPA-AUT, BDI-II, PDQ-39, and QUIP scales than patients classified as N−, suggesting that plasma NfL levels are related to cognitive function, and motor and non-motor symptoms.In the high-risk participants, although no significant differences were observed in cognitive function, or motor or non-motor symptoms between those classified as N+ and N−, participants classified as N+ had a higher rate of abnormal MIBG findings than those classified as N−, suggesting that plasma NfL indicates aSyn-induced neurodegeneration, particularly its peripheral involvement, at the prodromal phase.However, as NfL levels may be elevated in various neurodegenerative diseases, longitudinal observations are necessary to confirm phenoconversion and ascertain that the NfL elevation in the high-risk group is indeed caused by aSyn pathology.
Previous studies demonstrated that CSF aSyn is decreased in patients with PD 22 .However, results for studies on plasma aSyn levels have been inconsistent, possibly because plasma aSyn levels can be affected by contamination with red blood cells in which aSyn is abundant 23 .In the present study, we attempted to correct aSyn for hemoglobin levels.The plasma aSyn/Hb ratio was significantly decreased in the PD group compared with that in the high-risk and DLB groups and significantly elevated in the DLB group compared with that in the low-risk group.This inconsistent result indicates that plasma aSyn measurement via Simoa may have limitations, and techniques, such as real-time quaking-induced conversion (RT-QUIC), may be necessary 24 .
This study has some limitations.First, the sample size was small, and discrepancies in age and sex ratio among the groups were present.Therefore, the results of this study may not be generalizable and need to be   validated with a larger sample.Second, high-risk participants were selected based on a questionnaire survey on prodromal symptoms, and phenoconversion is yet to be confirmed.Further longitudinal studies are needed to confirm the precise risk of developing LBD in these individuals.Third, diagnoses of PD and DLB were based on clinical evaluations rather than neuropathological confirmation.Fourth, AT(N) profile was determined only by plasma biomarkers, and no PET or CSF studies were performed.Moreover, given the lack of neuropathological evaluations, attributing the changes in AD-related plasma biomarkers solely to AD pathology may overestimate the specificity of these biomarkers.Fifth, the cross-sectional nature of our study limits our ability to establish causal relationships or determine the temporal sequence of biomarker changes and symptom onset.Longitudinal studies are needed to achieve better understanding of the biomarker change in the prodromal phase of LBD.
In conclusion, our study demonstrated that comorbid AD neuropathology is present at the symptomatic phase of LBD.In PD, plasma Aβ composite was associated with general cognitive function, plasma p-tau181 with motor function and non-motor symptoms, and plasma NfL with cognitive and motor functions and non-motor symptoms.In addition, the elevated plasma NfL levels in the high-risk group, despite the absence of changes in AD-related plasma biomarkers, suggested the potential of plasma NfL as a biomarker to detect aSyn-induced neurodegeneration in the prodromal phase of LBD.

Study design and participants
The Nagoya-Takayama preclinical/prodromal Lewy body disease (NaT-PROBE) study is a prospective, longitudinal, multi-center, community-

ap
values determined by pairwise comparisons using Fisher's exact test with Benjamini-Hochberg correction.b p values determined by a one-way ANOVA with Tukey post-hoc test.

Table 1 |
Background characteristics of the participants Data represent the mean (standard deviation) or value (%).PD Parkinson's disease, DLB Dementia with Lewy bodies, DaT dopamine transporter, MIBG metaiodobenzylguanidine, MoCA-J the Japanese version of the Montreal Cognitive Assessment, LEDD Levodopa equivalent daily dose, MDS-UPDRS Movement Disorder Society-Unified Parkinson's Disease Rating Scale, SCOPA-AUT the Japanese version of the Scale for Outcomes in Parkinson's disease for Autonomic Symptoms, SAOQ Self-administered Odor Question, RBDSQ RBD screening scale, BDI-II Beck Depression Inventory-Second Edition, ESS Epworth Sleepiness Scale, PDQ-39 Parkinson's Disease Questionnaire-39, QUIP Questionnaire for Impulsive-Compulsive Disorders in Parkinson's disease.

Table 3 |
Clinical characteristics of the patients with Parkinson's disease grouped by A/T/N profiles

Table 4 |
2aT dopamine transporter, MIBG metaiodobenzylguanidine, MoCA-J the Japanese version of the Montreal Cognitive Assessment, LEDD Levodopa equivalent daily dose, MDS-UPDRS Movement Disorder Society-Unified Parkinson's Disease Rating Scale, SCOPA-AUT the Japanese version of the Scale for Outcomes in Parkinson's disease for Autonomic Symptoms, SAOQ Selfadministered Odor Question, RBDSQ RBD screening scale, BDI-II Beck Depression Inventory-Second Edition, ESS Epworth Sleepiness Scale, PDQ-39 Parkinson's Disease Questionnaire-39, QUIP Questionnaire for Impulsive-Compulsive Disorders in Parkinson's disease, Aβ amyloid-beta, p-tau181 phosphorylated tau 181, NfL neurofilament light chain, aSyn alpha-synuclein.A− Aβ composite < 0.376, A+ Aβ composite ≥ 0.376, T− log 10 (p-tau181) < 0.374, T+ log 10 (p-tau181) ≥ 0.374, N− log 10 (NfL) < 1.65, N+, log 10 (NfL) ≥ 1.65.valuesdeterminedbyanalysis of covariance (ANCOVA) adjusted for age and sex.dOne patient with PD could not complete the Stroop test.Two patients with PD could not complete the Stroop test.basedcohortstudycoordinated by the Nagoya University School of Medicine.Between March 2017 and January 2023, healthy individuals aged ≥50 years who visited the Kumiai Kosei Hospital, Daido Clinic, or Chutoen General Medical Center, in Japan, for their annual health checkup were surveyed using the following questionnaires: the Japanese version of the Scale for Outcomes in Parkinson's disease for Autonomic Symptoms (SCOPA-AUT); the Self-administered Odor Question (SAOQ); the RBD screening scale (RBDSQ); the Beck Depression Inventory-Second Edition (BDI-II); the Epworth Sleepiness Scale (ESS); and the Physical Activity Scale for the Elderly (PASE)3.Based on the results of our previous study 2 , 82 and 37 consecutive participants with ≥2 abnormal scores (high-risk group) and no abnormalities (low-risk group), respectively, in the SCOPA-AUT, SAOQ, and RBDSQ scales were enrolled in the present study.The cut-off value for identifying the high-risk group was 10, 90.0%, and 5 for the SCOPA-AUT, SAOQ, and RBDSQ scales, respectively2.In addition, patients with PD and DLB who visited Nagoya University Hospital, Kumiai Kosei Hospital, and the National Center for Geriatrics and Gerontology between March 2017 and January 2023 Clinical characteristics of the high-risk subjects grouped by A/T/N profiles Data represent the mean (standard deviation) or value (%).DaT dopamine transporter, MIBG metaiodobenzylguanidine, MoCA-J the Japanese version of the Montreal Cognitive Assessment, MDS-UPDRS Movement Disorder Society-Unified Parkinson's Disease Rating Scale, OSIT-J the odor stick identification test for Japanese, CVRR coefficient of variation of RR intervals, SCOPA-AUT the Japanese version of the Scale for Outcomes in Parkinson's disease for Autonomic Symptoms, SAOQ Self-administered Odor Question, RBDSQ RBD screening scale, BDI-II Beck Depression Inventory-Second Edition, ESS Epworth Sleepiness Scale, PDQ-39 Parkinson's Disease Questionnaire-39, QUIP Questionnaire for Impulsive-Compulsive Disorders in Parkinson's disease, Aβ amyloid-beta, p-tau181 phosphorylated tau 181, NfL neurofilament light chain, aSyn alphasynuclein.A− Aβ composite < 0.376, A+ Aβ composite ≥ 0.376, T− log 10 (p-tau181) <0.374, T+ log 10 (p-tau181) ≥0.374, N− log 10 (NfL) <1.65, N+ log 10 (NfL) ≥1.65.
a p values determined by Fisher's exact test.bp values determined by Student's t test.cp e a p values determined by Fisher's exact test.bp values determined by Student's t test.cp values determined by analysis of covariance (ANCOVA) adjusted for age and sex.