Journal of Affective Disorders 343 (2023) 144–152
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Journal of Affective Disorders
journal homepage: www.elsevier.com/locate/jad
Research paper
The association of serum neurofilament light chains with early symptoms
related to Parkinson’s disease: A cross-sectional study
Xueting Wang, Xin Yang, Weifeng He, Xin Song, Gaoman Zhang, Piye Niu, Tian Chen *
Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China
Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Parkinson’s disease
Neurofilament light chain
Early PD-related symptoms
Neurofilament light chains (NfL), released with neural axon injury, is considered as a potential biomarker for
Parkinson’s disease (PD). The relationship between NfL and PD has been studied mainly in diagnosed patients.
Few large-scale studies analyze the association between NfL levels and multiple non-motor symptoms linked to
early PD in the general population. Therefore, this study aims to determine the association of NfL with early
symptoms of PD, and effectively respond to the development of early symptoms of PD. We examined the rela­
tionship between serum NfL and early non-motor symptoms of PD (smell dysfunction, sleep problems, cognitive
function) and serum Klotho levels in the general population using data from the 2013–2014 National Health and
Nutrition Examination Survey (NHANES). The relationship between serum NfL and early symptoms of PD in
1125 participants was analyzed by multiple linear regression and logistic regression models. The results showed
a significant association between serum NfL and early symptoms of PD. There was a significant positive corre­
lation between NfL and smell dysfunction, short sleep and long sleep. There was a significant negative correlation
between NfL and Klotho levels and cognitive function test results. Further, we observed gender and age differ­
ences in the association of NfL with early symptoms of PD. Our study demonstrate that elevated serum NfL levels
are positively associated with an increased risk of early PD-related symptoms, suggesting that serum NfL can be a
promising biomarker for early PD.
1. Introduction
Parkinson’s disease (PD) is currently one of the fastest growing brain
disease in the world, with nearly one million people already living with
the disease in the U.S. (Chakraborty et al., 2020; Wijeratne and Fox,
2021). PD is a complex neurological disease caused by the loss of
dopaminergic neurons in the brain, with a wide range of symptoms, such
as motor and cognitive impairment (Simon et al., 2020). The long
duration of the disease, continuous deterioration of the disease symp­
toms and side effects of the treatment drugs result in a poor quality of life
for people with PD, which bring enormous stress and financial burden to
the lives of families and society (Chakraborty et al., 2020; Yang et al.,
2020). At present, PD is diagnosed on the basis of clinical imaging and
neurological function, combined with the history of the patient. In fact,
when patients develop motor symptoms, the loss of dopaminergic neu­
rons in the brain may be more than half and the neurological damage
may already be severe (Pagan, 2012). Therefore, early detection and
prevention are critical to the treatment of PD.
Studies have shown that olfactory dysfunction, sleep problems,
cognitive function, constipation and upright hypotension are early
events in the onset of PD (Schapira et al., 2017). However, these
symptoms occur relatively mildly and are not easily observed in most
cases (Xie and Hu, 2022). How to detect and diagnose early symptoms in
time is the major challenge in the early diagnosis and prevention of PD.
Currently, there are fewer reports of markers associated with early nonmotor symptoms of PD. Therefore, further search for biomarkers asso­
ciated with early non-motor symptoms of PD is needed.
Neurofilament light chain (NfL) is identified as a promising
biomarker for central nervous system diseases (Yuan and Nixon, 2021).
NfL is a neuron-specific component of the axonal cytoskeleton and
abundantly express in the axons of neurons (Gaetani et al., 2019). At
normal physiological levels, NfL is released from axons into the inter­
stitial space at low levels. When central nervous system diseases such as
inflammation or neural injury cause axonal damage, this can lead to a
* Corresponding author at: Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069,
China; Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China.
E-mail address: chentian@ccmu.edu.cn (T. Chen).
https://doi.org/10.1016/j.jad.2023.10.014
Received 2 July 2023; Received in revised form 2 September 2023; Accepted 4 October 2023
Available online 5 October 2023
0165-0327/© 2023 Elsevier B.V. All rights reserved.
X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
significant release of NfL (Disanto et al., 2017). The study has shown
that cerebrospinal fluid and serum NfL levels are higher in the PD group
than those in the healthy control population and that elevated NfL levels
can predict a more rapid progression of motor decline in PD patients (Liu
et al., 2022). A cross-sectional study has assessed the association be­
tween serum NfL and the Montreal Cognitive Assessment (MoCA) results
and has found that serum NfL levels are associated with cognitive
decline in patients with PD (Mao et al., 2023). In patients with early PD,
orthostatic hypotension is independently associated with higher plasma
NfL levels (Park et al., 2021). The above studies demonstrate the po­
tential of NfL as a marker of PD progression. However, there are few
existing studies on the association of NfL with various non-motor
symptoms (e.g., sleep problems, smell dysfunction) in early PD.
In addition, studies have shown that Klotho has a very important
function in nervous system diseases (Dubal et al., 2015; Kuang et al.,
2017). Klotho proteins are essential components of the endocrine
fibroblast growth factor (FGF) receptor complex and are highly
expressed in the kidney and choroid plexus (Bian et al., 2015; Kuro,
2019). Klotho has various physiological functions, including antiinflammatory effects and autophagy regulation (Xu and Sun, 2015).
Mice with Klotho deficiency can develop oxidative stress, neurological
damage, and motor and cognitive deficits (Anamizu et al., 2005; Kuro-o
et al., 1997; Nagai et al., 2003; Vo et al., 2018). Intestinal inflammation
may be a very early stage of PD that can ascend through the vagus nerve
to the central nervous system, and it is important in the mechanism of
PD development (Grillo et al., 2022). Klotho is a potent biomarker to
characterize the inflammatory state (Wu and Chen, 2022). When in­
testinal inflammation occur, it is accompanied by a downregulation of
Klotho levels (Thurston et al., 2010). The above studies indicate that
Klotho have a very critical role in the pathological mechanisms of PD.
This suggests that we can further explore the role of NfL in the early
stages of PD by examining the association between NfL and Klotho.
Based on the role of NfL in neural injury and the fact that early nonmotor symptoms of PD are not easily detected, we hypothesis that NfL
can be considered as a potential biomarker for early non-motor symp­
toms of PD. Therefore, this study investigate the association between
serum NfL and early PD-related symptoms (smell dysfunction, sleep
problems, cognitive function) and Klotho levels in the general popula­
tion using the National Health and Nutrition Examination Survey
(NHANES) dataset. As the occurrence of PD present differently by
gender and age (Vaidya et al., 2021), a stratified study by age and
gender is also conducted to further explore the association between NfL
and early symptoms of PD. This study demonstrate the potential of NfL
in predicting early PD-related symptoms and provide new epidemio­
logical data for the diagnosis of PD.
problems. A total of 1206 subjects had complete data for the four PDrelated indicators mentioned above, and after further exclusion of sub­
jects with incomplete covariate data, 1125 participants were finally
included in this study. As only adults over 40 years of age had serum
Klotho measurements and smell function test results, the subjects in this
study were adults over 40 years of age. In addition, NHANES data pro­
vided cognitive test data for people over the age of 60, and 450 subjects
in this study had complete cognitive function test data and covariates.
The specific screening process for the study population was shown in
Fig. S1.
2.2. Serum NfL measurement
NfL measurements were carried out on a fully automated Attelica
immunoassay system using a highly sensitive and high throughput
acridinium-ester (AE) immunoassay (Lee et al., 2022). Specifically, the
samples were first incubated with an AE-labelled antibody that binded
to the NfL antigen. Next, paramagnetic particles (PMP) coated with the
capture antibody were added to the sample to form an antigenic com­
plex bound to the AE-labelled antibody and PMP. The unbound AElabelled antibody was then separated and removed, followed by the
addition of acid and base to initiate chemiluminescence and measure­
ment of light emission. The lower limit of quantification (LLOQ) for this
assay was 3.9 pg/mL, determined by replication testing of low concen­
tration NfL samples, and the upper limit of quantification (ULOQ) was
500 pg/mL. The LLOQ was defined as a concentration where the coef­
ficient of variation (CV) was less than or equal to 20 %.
2.3. Serum α-Klotho measurement
Klotho concentrations were performed by a commercial ELISA kit
produced by IBL International, Japan. Assay samples were original
serum samples from participants aged 40–79 years in the NHANES
2013–2014 cycle, and fresh frozen serum samples stored at − 80 ◦ C were
shipped from the Centers for Disease Control and Prevention to the
Northwest Lipid Metabolism and Diabetes Research Laboratory,
Department of Metabolism, Endocrinology and Nutrition, University of
Washington, and assayed for analysis during 2019–2020. The analysis
was repeated for the samples and the average of the two values was used
for the calculation of the final values. The relative signals of the assay
standard curves and calibrator concentrations were always within the
manufacturer’s specified standard range and the sensitivity of the assay
obtained was calculated to be 4.33 pg/mL. Two samples with very high
and high Klotho concentrations were used at different dilutions to assess
the assay linearity. Plots of expected versus obtained values showed
excellent linearity in the assay measurement range.
2. Method
2.4. Identification of early symptoms of PD
2.1. Study design and participants
For the detection of the olfactory function, we obtained data from the
examination data for the smell test (The 8-Item Odor Identification
Test). The odor test consisted of four food-related odors and four nonfood-related odors, the latter including smoke and natural gas. The
eight odors were released by scratching the test paper and presented in a
fixed order: chocolate, strawberry, smoke, leather, soap, grape, onion
and natural gas. Above each odor strip was a list of four possible re­
sponses, and the participant was asked to choose one of the four options
presented. If the participant indicated that he/she could not smell any
odor when he/she smelt it, he/she was required to select one of the four
choices as the answer. Those who were unable to correctly identify six
and above were considered to have olfactory dysfunction (Zheng et al.,
2020).
For the sleep question, we categorized the sleep time according to the
answer to the question “ How much sleep do you get (hours)?”, including
short sleep (≤6 h), mid-range sleep (7–8 h) and long sleep (≥9 h)
(Cavallino et al., 2022). We also combined the subjective reporting of
The National Health and Nutrition Examination Survey (NHANES,
https://www.cdc.gov/nchs/nhanes) was a program of the National
Center for Health Statistics (NCHS) that focused on assessing the phys­
ical health and nutritional status of adults and children in the United
States. It was collected from a nationally representative sample of the
United States and consisted primarily of demographic data, screening
data, laboratory data and questionnaire data. As only the 2013–2014
NHANES data currently contained NfL measurements, data from this
year were obtained for this study. Informed consent was obtained from
all participants, and specific information on the data procedures and
content of NHANES could be found on the website (https://www.cdc.
gov/nchs/nhanes).
A total of 10,175 subjects participated in NHANES from 2013 to
2014, of which 2085 and 2767 subjects had serum NfL or Klotho mea­
surements respectively, 3708 subjects underwent smell function testing
and 6464 subjects answered questions related to sleep time and
145
X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
sleep problems by participants and whether a doctor had diagnosed
having a sleep disorder to determine whether participants had sleep
problems.
Cognitive functioning was assessed through a combination of three
areas of testing, the CERAD Word Learning subtest (CERAD W-L), the
Animal Fluency Test (AFT) and the Digit Symbol Substitution Test
(DSST). The CERAD W-L assessed immediate and delayed learning of
new linguistic information, which included the Immediate Recall Test
(IRT) and the Delayed Recall Test (DRT). IRT involved three consecutive
learning trials in which participants were instructed to read aloud 10
unrelated words, one at a time, during the presentation. Immediately
after word presentation, participants recalled as many words as possible.
The order of the 10 words changed on each trial, and the total score for
all three trials ranged from 0 to 30. The DRT was administered after the
other two cognitive tests (AFT and DSST) completed (approximately
8–10 min from the beginning of the word learning trial). The AFT was
used to check categorical verbal fluency and participants were asked to
name as many animals as possible in 1 min and a point was given for
each named animal. The DSST was the performance module of the
Wechsler Adult Intelligence Scale (WAIS III) and relied on processing
speed, sustained attention and working memory. Participants were
asked to copy the symbols paired with the number provided at the top of
the exercise sheet. The score was the total number of correct matches
within 2 min. Lower scores indicated poorer cognitive functioning in
each aspect of the cognitive functioning test.
3. Result
3.1. Characteristics of study participants grouped by age
The demographic characteristics of the participants were shown in
Table 1. Among all participants, the mean (SD) for serum cotinine levels
was 60.28 (137.47) ng/mL and 53.87 % were female. The most partic­
ipants were Non-Hispanic White (44.80 %), and had a BMI over 30 kg/
m2 (39.64 %), an education level of Some College or AA degree (31.02
%), an alcohol consumption (71.91 %) and a survey cycle between
November 1 through April 30 (50.49 %). After stratification based on
age, there were 655 people younger than 60 years and 470 people older
than 60 years, and both had similar proportions for gender, race, BMI,
alcohol status and six month time period when surveyed. Education
level and serum cotinine levels were significantly higher in adults (<60
years) than those in older adults (≥60 years). We further stratified the
demographic information of the participants by gender (Table S1), and
men had significantly higher alcohol status and serum cotinine levels
than women, while women had a relatively high BMI.
Table 1
Characteristics of participants grouped by age.
Characteristics
2.5. Covariates
Mean (SD) or n (%)
Gender
We identified covariates associated with PD based on previous
studies, including gender, age (years), race/ethnicity (Mexican Amer­
ican, other Hispanic, non-Hispanic White, non-Hispanic Black and Other
races), education (less than 9th grade, 9-11th grade, high school grade,
some college or AA degree and college graduate or above), six month
time period when surveyed (November 1 through April 30, May 1
through October 31). BMI data were classified into 3 categories
including ≤25, 25.1–29.9, and ≥ 30 kg/m2. Alcohol status was deter­
mined based on subjects having at least 12 alcoholic drinks per year.
Exposure to environmental tobacco smoke is usually estimated using
questionnaires, but they are unreliable. Biomarkers are the most
commonly used objective method for determining nicotine exposure.
Studies have shown that the best biomarker of nicotine exposure is co­
tinine (Florescu et al., 2007). Therefore, serum cotinine levels were used
to assess smoke exposure.
Men
Women
Race
Mexican American
Other Hispanic
Non-Hispanic White
Non-Hispanic Black
Other races
BMI (kg/m2)
≤25
25.1–29.9
2.6. Statistical analysis
≥30
Education
Less than 9th grade
Student’s t-test or Mann-Whitney test and chi-square test were used
for statistical analysis of differences between the two groups for
continuous and categorical variables, respectively. NfL and Klotho
concentrations were log-transformed and used for subsequent analyses.
The association of NfL with Klotho or cognitive test results was exam­
ined by spearman analysis. In order to examine the association between
NfL and indicators of early PD symptoms, the association between NfL
and Klotho or cognitive test results was determined by multiple linear
regression. Binary logistic regression analysis was used to determine the
association between NfL and smell dysfunction and subjective reports of
sleep problems. Multinomial logistic regression analysis was used to
determine the association between NfL and sleep duration classification.
In above analyses, age, gender, race/ethnicity, education level, BMI
category, alcohol status, serum cotinine levels and the six-month time
period at the time of the survey were controlled. Further stratified an­
alyses were conducted according to gender and age. Data were analyzed
using r (4.1.2). Two-sided p-values <0.05 were statistically significant.
9-11th grade
High school grade
Some college or AA
degree
College graduate or
above
Alcohol status
Yes
No
Cotinine (ng/mL)
Six month time period
when surveyed
November 1 through
April 30
May 1 through
October 31
BMI, body mass index.
146
Total (n =
1125)
<60 years (n
= 655)
≥60 years (n
= 470)
519 (46.13
%)
606 (53.87
%)
302 (46.11
%)
353 (53.89
%)
217 (46.17
%)
253 (53.83
%)
162
%)
110
%)
504
%)
198
%)
151
%)
103 (15.73
%)
59 (12.55 %)
65 (9.92 %)
45 (9.57 %)
286 (43.66
%)
107 (16.34
%)
218 (46.38
%)
94 (14.35 %)
57 (12.13 %)
282 (25.07
%)
397 (35.29
%)
446 (39.64
%)
156 (23.82
%)
227 (34.66
%)
272 (41.53
%)
126 (26.81
%)
170 (36.17
%)
174 (37.02
%)
95 (8.44 %)
154 (13.69
%)
219 (19.47
%)
349 (31.02
%)
308 (27.38
%)
41 (6.26 %)
54 (11.49 %)
86 (13.13 %)
68 (14.47 %)
118 (18.02
%)
208 (31.76
%)
202 (30.84
%)
101 (21.49
%)
141 (30.00
%)
106 (22.55
%)
809 (71.91
%)
316 (28.09
%)
60.28
(137.47)
483 (73.74
%)
172 (26.26
%)
68.54
(141.32)
326 (69.36
%)
144 (30.64
%)
48.78
(131.20)
568 (50.49
%)
557 (49.51
%)
337 (51.45
%)
318 (48.55
%)
231 (49.15
%)
239 (50.85
%)
(14.40
(9.78
(44.80
(17.60
(13.42
pvalue
0.983
0.305
91 (19.36 %)
0.279
0.001
0.107
0.014
0.446
X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
3.2. NfL levels and PD-related early indicators of grouped by age
Table 2 showed the serum NfL and Klotho concentrations, the results
of the cognitive tests and the percentage of smell dysfunction and sleep
problems. Among all participants, the mean (SD) for serum NfL and
Klotho concentrations was 19.74 (21.56) pg/ml and 861.24 (284.73)
pg/ml respectively and 142 individuals (12.62 %) were smell dysfunc­
tion. For sleep time, 37.78 % were in short sleep, 7.47 % were in long
sleep and 33.16 % subjectively reported having sleep problems. The
mean (SD) for the four cognitive function test results (IRT, DRT, AFT,
DSST) were 20.61 (4.31), 6.69 (2.13), 17.40 (5.75) and 49.02 (17.45)
respectively. After stratification according to age, serum NfL levels, and
the proportion of people with long sleep and smell dysfunction were
significantly higher in older adults (≥60 years), while adults younger
than 60 years had a higher proportion of short sleep time. Table S2
showed the results further stratified by gender. Compared to men,
women had higher Klotho levels and cognitive test results (IRT, DSST),
more sleep problems, and fewer smell dysfunction.
Fig. 1. Spearman’s correlation between log-transformed serum NfL and Klotho
levels. NfL, neurofilament light chain.
3.3. Regression analysis between NfL levels and Klotho levels
As shown in Fig. 1, NfL levels and Klotho levels in serum were
significantly negatively correlated (r = − 0.084, p = 0.0048). Then we
carried out the linear regression and presented the regression results for
serum NfL levels with Klotho levels and adjusted variables (Table 3,
Table S3). Among overall participants, serum levels of Klotho showed a
significant negative correlation with NfL levels (unadjusted model: β
(95 % CI) = − 0.047 (− 0.077, − 0.018), p = 0.002; adjusted model: β (95
% CI) = − 0.032 (− 0.064, 0.000), p = 0.047). We further explored the
association between NfL and Klotho levels classified by age and gender
(Table 3, Fig. S2A). In the female and elderly groups, the association
Table 3
Associations between NfL levels and Klotho levels.
Klotho
Unadjusted
β (95 % CI)
All subjects
a
Gender
Men
Table 2
Characterization of serum NfL levels and early indicators of PD after age
stratification.
Characteristics
Total (n =
1125)
<60 years (n
= 655)
≥60 years (n
= 470)
19.74
(21.56)
861.24
(284.73)
15.85
(14.49)
873.18
(279.04)
25.16
(27.76)
844.61
(291.96)
Women
c
Age
<60
years
≥60
years
p-value
Mean (SD) or n (%)
NfL (pg/ml)
Klotho (pg/ml)
Smell dysfunction
Yes
No
Sleep duration
Short sleep (≤6 h/
night)
Mid-range sleep
(7–8 h/night)
Long sleep (≥9 h/
night)
Sleep problems
Yes
No
Cognitive function
indicatorsa
IRT
DRT
AFT
DSST
142 (12.62
%)
983 (87.38
%)
84 (17.87 %)
597 (91.15
%)
386 (82.13
%)
425 (37.78
%)
616 (54.76
%)
278 (42.44
%)
339 (51.76
%)
147 (31.28
%)
277 (58.94
%)
84 (7.47 %)
38 (5.80 %)
46 (9.79 %)
373 (33.16
%)
753 (66.93
%)
216 (32.98
%)
439 (67.02
%)
157 (33.40
%)
313 (66.60
%)
–
–
–
–
–
–
–
–
20.61 (4.31)
6.69 (2.13)
17.40 (5.75)
49.02
(17.45)
− 0.047 (− 0.077,
− 0.018)
− 0.024 (− 0.066,
0.018)
− 0.063 (− 0.104,
− 0.022)
− 0.043 (− 0.083,
− 0.003)
− 0.041 (− 0.092,
0.011)
0.002
0.258
0.002
0.034
0.122
β (95 % CI)
− 0.032 (− 0.064,
0.000)
−
−
−
−
0.017 (− 0.062,
0.029)
0.046 (− 0.091,
0.001)
− 0.035 (− 0.075,
0.005)
− 0.035 (− 0.087,
0.018)
pvalue
0.047
0.471
0.044
0.090
0.192
a
Adjusted for gender, age, race, BMI, Education, Alcohol status, Six month
time period when surveyed and serum cotinine levels.
b
Adjusted for age, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
c
Adjusted for gender, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
<0.001
0.055
<0.001
58 (8.85 %)
b
Adjusted
pvalue
showed a significant negative correlation (female: r = − 0.11, p =
0.0079; older adults: r = − 0.092, p = 0.046). In women, NfL and Klotho
were significantly negatively correlated (unadjusted model: β (95 % CI)
= − 0.063 (− 0.104, − 0.022), p = 0.002; adjusted model: β (95 % CI) =
− 0.046 (− 0.091, − 0.001), p = 0.044). There was also a significant
negative association between NfL and Klotho in the unadjusted model in
the <60 years group (β (95 % CI) = − 0.043 (− 0.083, − 0.003), p =
0.034). However, no significant associations were observed by regres­
sion analysis in men and older adults.
<0.001
0.881
3.4. Regression analysis between NfL levels and smell dysfunction
As shown in Fig. S3A, the concentration of serum NfL was higher in
those with smell dysfunction. And this phenomenon was more signifi­
cant in males (Fig. S3D). Then, the association between serum NfL levels
and smell dysfunction was demonstrated using a binary logistic regres­
sion model (Table 4). Table S4 listed the variables that were adjusted for
during the regression. There was a significant positive association be­
tween serum NfL levels and smell dysfunction in all populations
NfL, neurofilament light chain. IRT, Immediate Recall Test. DRT, Delayed Recall
Test. AFT, Animal Fluency Test. DSST, Symbol Substitution Test.
a
Participants with cognitive functioning indicators were all >60 years old and
there were 450 individuals in total.
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X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
Table 4
Associations between NfL levels and smell dysfunction.
Table 5
Associations between NfL levels and sleep duration and sleep problems.
Smell dysfunction
Unadjusted
Unadjusted
All subjects a
b
Gender
Men
Women
Age c
<60 years
≥60 years
Adjusted
OR (95 % CI)
p-value
OR (95 % CI)
pvalue
1.677 (1.277, 2.198)
<0.001
1.449 (1.056, 1.972)
0.020
1.889 (1.303, 2.747)
1.409 (0.930, 2.104)
<0.001
0.098
1.733 (1.131, 2.645)
1.210 (0.718, 1.980)
0.011
0.461
1.273 (0.808, 1.960)
1.549 (1.026, 2.329)
0.285
0.035
1.301 (0.816, 2.031)
1.457 (0.925, 2.267)
0.256
0.098
Adjusted
OR (95 % CI)
p-value
OR (95 % CI)
p-value
1.031 (0.840,
1.266)
0.772
1.268 (1.007,
1.596)
0.043
Short sleep (≤6 h/
night)
All subjects a
Gender b
Men
Women
Age c
a
Adjusted for gender, age, race, BMI, Education, Alcohol status, Six month
time period when surveyed and serum cotinine levels.
b
Adjusted for age, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
c
Adjusted for gender, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
<60 years
≥60 years
Long sleep (≥9 h/
night)
All subjects a
(unadjusted model: OR (95 % CI) = 1.677 (1.277, 2.198), p < 0.001;
adjusted model: OR (95 % CI) = 1.449 (1.056, 1.972), p = 0.020).
Similar regression model results were also found in men (unadjusted
model: OR (95 % CI) = 1.889 (1.303, 2.747), p < 0.001; adjusted model:
OR (95 % CI) = 1.733 (1.131, 2.645), p = 0.011). Besides, a positive
association was found in the unadjusted model for the elderly (unad­
justed model: OR (95 % CI) = 1.549 (1.026, 2.329), p = 0.035). How­
ever, this significant association disappeared in women and those
younger than 60 years.
Gender
b
Men
Women
Age
c
<60 years
≥60 years
Sleep problems
3.5. Regression analysis between NfL levels and sleep time and sleep
problems
All subjects a
Gender
We first examined NfL concentrations in subgroups with different
sleep time and sleep problems. We found that NfL concentrations were
significantly higher in those with longer sleep durations and were more
pronounced in the elderly (Fig. S3C, F). And NfL concentrations were
significantly higher in men with sleep problems (Fig. S3B, E). Table 5
demonstrated the association between NfL levels and sleep duration and
sleep problems. Table S5 showed the adjusted variables in the re­
gressions. In all participants, NfL levels were found to be positively
associated with short sleep (adjusted model: OR (95 % CI) = 1.268
(1.007, 1.596), p = 0.043) and long sleep (unadjusted model: OR (95 %
CI) = 1.968 (1.402, 2.765), p < 0.001; adjusted model: OR (95 % CI) =
1.996 (1.365, 2.918), p < 0.001). The relationship between NfL levels
and long sleep could be observed in men, women (unadjusted model)
and older adults. However, no significant association between NfL levels
and sleep problems was observed.
b
Men
Women
Age
c
<60 years
≥60 years
1.007 (0.741,
1.368)
1.071 (0.811,
1.414)
1.181 (0.897,
1.555)
1.261 (0.866,
1.835)
1.968 (1.402,
2.765)
2.430 (1.475,
4.004)
1.664 (1.038,
2.668)
1.668 (0.980,
2.839)
2.307 (1.375,
3.869)
1.176 (0.961,
1.437)
1.276 (0.940,
1.732)
1.142 (0.872,
1.497)
1.113 (0.841,
1.468)
1.346 (0.952,
1.907)
0.966
0.630
0.237
0.227
<0.001
<0.001
0.034
0.059
0.002
0.114
0.117
0.333
0.451
0.092
1.196 (0.854,
1.674)
1.306 (0.947,
1.801)
1.229 (0.922,
1.639)
1.359 (0.911,
2.026)
1.996 (1.365,
2.918)
2.288 (1.316,
3.979)
1.677 (0.983,
2.861)
1.711 (0.962,
3.041)
2.291 (1.340,
3.918)
1.160 (0.926,
1.452)
1.116 (0.793,
1.569)
1.200 (0.881,
1.634)
1.054 (0.782,
1.414)
1.353 (0.939,
1.959)
0.298
0.104
0.160
0.133
<0.001
0.003
0.058
0.067
0.002
0.195
0.526
0.245
0.728
0.106
a
Adjusted for gender, age, race, BMI, Education, Alcohol status, Six month
time period when surveyed and serum cotinine levels.
b
Adjusted for age, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
c
Adjusted for gender, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
test scores. The association between NfL levels and AFT was more sig­
nificant in women, while the association with DSST was more pro­
nounced in men (Fig. S2C, E, Table 6). In addition, we also found a
significant negative correlation between NfL and DRT, IRT in women
(Fig. S2B, D).
In addition, according to previous reports (Bornhorst et al., 2022; Hu
et al., 2021; Lerche et al., 2020), there was a large association between
NfL and cognitive function with age, we further analyzed the association
between NfL levels and four cognitive tests by multiple linear regression
without age as a covariate (Table S7). We found that NfL levels and
scores on four cognitive tests both showed stronger significant negative
correlations, and this phenomenon was more pronounced in females.
3.6. Regression analysis between NfL levels and scores on four cognitive
tests
We observed the association between the serum NfL levels and the
scores on the four cognitive tests, all of which were significantly nega­
tively correlated (Fig. 2). As shown in Table 6, further results from
multiple linear regression revealed a significant negative association
between NfL levels and IRT (unadjusted model: β (95 % CI) = − 1.077
(− 1.801, − 0.353), p = 0.004; adjusted model: β (95 % CI) = − 0.724
(− 1.445, − 0.004), p = 0.049), DRT (unadjusted model: β (95 % CI) =
− 0.427 (− 0.785, − 0.069), p = 0.019), AFT (unadjusted model: β (95 %
CI) = − 1.537 (− 2.501, − 0.572), p = 0.002; adjusted model: β (95 % CI)
= − 1.060 (− 1.969, − 0.152), p = 0.022) and DSST (unadjusted model: β
(95 % CI) = − 5.707 (− 8.615, − 2.800), p < 0.001; adjusted model: β (95
% CI) = − 3.507 (− 5.769, − 1.244), p = 0.002). Table S6 presented the
results of the covariate regressions associated with the four cognitive
4. Discussion
NfL has already been shown to be elevated in some neurodegener­
ative diseases (Forgrave et al., 2019; Haji et al., 2022). The increased
levels of NfL may be associated with an increased risk of early non-motor
148
X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
Fig. 2. Spearman’s correlation between log-transformed serum NfL and scores on four cognitive tests. NfL, neurofilament light chain. IRT, Immediate Recall Test.
DRT, Delayed Recall Test. AFT, Animal Fluency Test. DSST, Symbol Substitution Test.
symptoms in PD. In this study, we found that serum NfL levels were
significantly and positively associated with smell dysfunction, short and
long sleep, and significantly negatively correlated with Klotho levels and
cognitive function. In conclusion, this study demonstrates a clear asso­
ciation between elevated serum NfL and the development of early PDrelated symptoms, suggesting that serum NfL may be a promising
biomarker as an early non-motor symptom of PD.
Olfactory dysfunction is thought to be an early symptom of some
neurodegenerative diseases. The olfactory bulb is the first line of defense
against pathogens or external environmental contact, and pathological
protein aggregates affect this area first compared to other areas,
damaging the nerve cells in this area and then transmitting to the brain
via the olfactory pathway (Marin et al., 2018; Rey et al., 2018). A
multicenter study has shown that 96.7 % of PD patients have significant
olfactory loss compared to younger controls, and 74.5 % of PD patients
still have olfactory loss after age-related adjustment (Haehner et al.,
2009). Despite this, studies have found that 72 % of people with PD are
unaware that they have an olfactory impairment until they are tested for
olfactory function (Doty et al., 1988). It is also difficult to quantify ol­
factory impairment clinically, and the actual condition of olfactory
impairment may not match the subjective symptoms, or may be influ­
enced by the external environment or other diseases (Park et al., 2018).
This study found higher concentrations of serum NfL in people with
olfactory dysfunction, suggesting that nerve cells in the olfactory region
were damaged in population, leading to reduced olfactory function. The
regression results showed a significant positive association between
serum NfL and olfactory impairment in the overall population, with or
without adjustment for relevant covariates. In combination with existing
studies, serum NfL may be a good predictor of olfactory impairment. We
also observed that the proportion of elderly people with olfactory
impairment was significantly higher than those younger than 65 years.
PD is an age-dependent neurodegenerative disease with an increasing
prevalence with age (Lenka et al., 2017). Aging can also lead to olfactory
disturbances, which may be related to changes in olfactory structures,
the development of nasal diseases and changes in the central brain areas
involved in olfactory processing (Doty and Kamath, 2014). We identified
that olfactory impairment was more severe in men, as previous studies
found (Mullol et al., 2012). This may be due to the higher number of
cells (both neurons and non-neurons) in the olfactory bulb in females
compared to males (Oliveira-Pinto et al., 2014).
Sleep problems is also one of the common early non-motor symptoms
of PD, mainly including rapid eye movement sleep behavior disorder,
sleep disorders and insomnia (Maggi et al., 2023). The NHANES data­
base does not contain specific causes or modalities of short sleep, and we
cannot further determine if it is a more relevant sleep problem for PD,
but we still get preliminary conclusions. After adjusting for relevant
variables, we found a significant positive correlation between NfL levels
and short sleep. Thus, elevated serum levels of NfL may be associated
with early symptoms of PD (difficulty falling asleep), but more studies
are needed to further determine the association of serum NfL with PDrelated sleep problems. Notably, we also found that NfL levels were
significantly higher in the long sleep group and regression results indi­
cated that NfL levels showed a significant positive correlation with long
sleep. This was more pronounced in men and older adults. An epide­
miological study has shown that both short and long sleep duration can
cause certain health disorders in older people, showing a U-shaped
trend, including depressive symptoms, increased body mass index and
amyloid, and cognitive decline (Winer et al., 2021). Studies have also
demonstrated that PD-related risk factors such as fatigue, immune
function, abnormal photoperiod or reduced health may lead to
149
X. Wang et al.
Journal of Affective Disorders 343 (2023) 144–152
after peripheral injection of lipopolysaccharide into the mice (Zhu et al.,
2018). Exogenous Klotho can decrease pro-inflammatory factors in Aβexposed cells, including nuclear factor-kB (NF-kB), interleukin-1β (IL1β) and tumor necrosis factor-α (TNF-α) (Sedighi et al., 2021). Our re­
sults found that NfL showed a significant negative correlation with
Klotho and the regression results yielded the same conclusion. This
suggest us that NfL may be a better indicator of the response to PDrelated neural injury. We also found that this phenomenon was more
pronounced in women. More disease-related immune and angiogenic
mediators are produced in the stools of women with PD, while no such
difference is found when comparing male PD patients with controls by
analyzing stools of PD patients and healthy people (Houser et al., 2018).
The above reflect that there are mechanistic differences in PD-related
symptoms by gender.
In fact, it has been well documented that NfL can be a biomarker of
cognitive decline in PD, and the same finding has been obtained in our
study. More importantly, we observed gender differences in cognitive
function. The female PD patients perform better on Symbolic Digit
Pattern Test, Verbal Fluency Test, and Overall Cognition on the MoCA,
but have worse visuospatial functioning (Lin et al., 2018). This suggest
that there may be different mechanisms of occurrence of cognitive
decline in different gender in PD, but the exact mechanism is still un­
known and need further study.
There are also some limitations in our study. Firstly, this is a crosssectional study and there is no way to obtain a causal association be­
tween NfL and early indicators related to PD. And this study cannot
obtain actual diagnostic data for PD. The association between serum NfL
and early PD-related markers may be due to early PD, but it may also be
due to other causes or diseases. Therefore, more epidemiological studies
or in vivo and in vitro experiments are needed. Next, NHANES collects
limited data. Some of the early symptoms associated with PD are
nonspecific and may occur in other neurological injury disorders. For
example, there are many forms or symptoms of sleep problems, and
NHANES only collects self-reported presence or absence of sleep prob­
lems and cannot measure the association between the type of sleep
disorder associated with PD and NfL. Finally, the population in this
study is U.S. adults aged 40 years and older and the conclusions need to
be generalized with caution if they need to be generalized to other age
groups or other countries.
Despite these limitations, our study is the first to examine the asso­
ciation between serum NfL and a variety of early PD-related symptoms
in the general population, extending the existing studies. The value of
our study aims to validate previous relevant findings and show that
serum NfL predicts an elevated risk for the development of clinical
symptoms associated with the early stages of PD. Serum samples are
more practical as biomarkers in clinical diagnosis than non-motor
symptoms that are not easily examined and detected in the early
stages of PD, facilitating early diagnosis and treatment of PD patients.
Table 6
Associations between NfL levels and scores on four cognitive tests.
Unadjusted
β (95 % CI)
IRT
All subjects
a
Gender b
Men
Women
DRT
All subjects
a
Gender
b
Men
Women
AFT
All subjects
a
Gender b
Men
Women
DSST
All subjects
a
Gender
b
Men
Women
− 1.077
(− 1.801–0.353)
− 0.881 (− 1.956,
0.195)
− 1.161 (− 2.137,
− 0.186)
− 0.427 (− 0.785,
− 0.069)
− 0.299 (− 0.832,
0.234)
− 0.514 (− 0.999,
− 0.028)
− 1.537 (− 2.501,
− 0.572)
− 1.314 (− 2.765,
0.137)
− 1.803 (− 3.099,
− 0.508)
− 5.707 (− 8.615,
− 2.800)
− 7.336 (− 11.424,
− 3.249)
− 3.916 (− 8.003,
0.171)
Adjusted
p-value
0.004
0.108
0.020
0.019
0.270
0.038
0.002
0.076
0.007
<0.001
<0.001
0.060
β (95 % CI)
− 0.724 (− 1.445,
− 0.004)
− 0.634 (− 1.687,
0.419)
− 0.979 (− 1.990,
0.033)
− 0.263 (− 0.620,
0.094)
− 0.095 (− 0.605,
0.414)
− 0.420 (− 0.932,
0.092)
− 1.060 (− 1.969,
− 0.152)
− 0.828 (− 2.231,
0.575)
− 1.367 (− 2.591,
− 0.143)
− 3.507 (− 5.769,
− 1.244)
− 4.744 (− 7.911,
− 1.576)
− 2.107 (− 5.384,
1.170)
pvalue
0.049
0.237
0.058
0.149
0.713
0.107
0.022
0.246
0.029
0.002
0.004
0.206
IRT, Immediate Recall Test. DRT, Delayed Recall Test. AFT, Animal Fluency
Test. DSST, Symbol Substitution Test.
a
Adjusted for gender, age, race, BMI, Education, Alcohol status, Six month
time period when surveyed and serum cotinine levels.
b
Adjusted for age, race, BMI, Education, Alcohol status, Six month time
period when surveyed and serum cotinine levels.
prolonged sleep, which can lead to an increased risk of death (Grandner
and Drummond, 2007). As for gender differences, studies have shown
that the prevalence of insomnia is about 1.5 times higher in women than
that in men, which may be related to physiological factors (e.g. estrogen,
menstruation, pregnancy) and psychological factors (e.g. anxiety, stress)
(Suh et al., 2018). This may explain the gender differences in the asso­
ciation between NfL and long sleep. Combined with the current study,
we think that NfL may be a better biomarker for sleep difficulties.
However, in this study, no association between NfL and self-reported
sleep problems was observed. This may be since there are too few
questions about sleep problems in the NHANES data, and more reliable
and accurate questions are needed to confirm the diagnosis of sleep
problems and investigate the symptoms of sleep problems associated
with PD.
Klotho is an anti-aging protein and its overexpression in mice can
prolong their lifespan (Kurosu et al., 2005). It has been found that Klotho
expression is decreased in a variety of central nervous system diseases
(Emami Aleagha et al., 2015; Semba et al., 2014; Teocchi et al., 2013).
The specific mechanisms of Klotho-mediated neuroprotection involve
stimulation of synaptic function, anti-oxidative stress effects and antiinflammatory effects (Hanson et al., 2021). The expression of several
pro-inflammatory factors is increased in the choroid plexus of Klotho
knockout transgenic mice, and this also cause activation of microglia
5. Conclusion
In conclusion, this study found that elevated serum NfL was signifi­
cantly associated with early PD-related symptoms and Klotho levels. We
observed that serum NfL levels were significantly positively correlated
with smell dysfunction, short and long sleep, and significantly nega­
tively correlated with Klotho levels and cognitive function. We also
found differences in the above associations between gender and age,
which might be related to the pathogenesis of PD. Future studies are
needed to confirm our findings and explore potential mechanisms.
Role of the funding source
This study was supported by the National Natural Science Founda­
tion of China (No. 82173489), and the Beijing Natural Science Foun­
dation (No. 7232234). The funder of the study had no role in study
design, data collection, data analysis, data interpretation, or writing of
150
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Journal of Affective Disorders 343 (2023) 144–152
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CRediT authorship contribution statement
Xueting Wang: Writing - Original Draft, Data analysis, Software. Xin
Yang: Investigation, Software. Weifeng He: Software. Xin Song: Inves­
tigation. Gaoman Zhang: Methodology. Piye Niu: Project administra­
tion. Tian Chen: Software, Writing - review & editing, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This study was supported by the National Natural Science Founda­
tion of China (No. 82173489), and the Beijing Natural Science Foun­
dation (No. 7232234).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jad.2023.10.014.
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