Oxidative stress plays a significant role in the pathogenesis of many diseases, including neurodegenerative diseases [
Traditionally, body fluid biomarkers, including cerebrospinal fluid (CSF), serum, or plasma, have been evaluated by immunoassay techniques. However, the accuracy of the results may be affected owing to the manual protocols involved, indicating the need for a more objective and precise method. The immunomagnetic reduction-based immunoassay (IMR) was introduced, and the application was validated to quantitatively measure the plasma A
Studies have reported alterations of both dopaminergic and nondopaminergic transmitter systems in the PD brain with different degrees of cognitive impairment [
Based on previous reports, the oxidative stress microenvironment affecting neurons could result in not only the generation of abnormal protein aggregates but also deficits in the microstructure of the brain, and possibly brain volume loss or neuron loss [
A total of 31 patients with no previous underlying neurological or psychological/psychiatric diseases, taking no neurological or psychotropic medication, were enrolled at the Neurology Outpatient Department in Kaohsiung Chang-Gung Memorial Hospital prospectively. All patients were with no contraindications for receiving magnetic resonance imaging (MRI) and wrote informed consents prior to participation in this study. All patients diagnosed with Parkinson’s disease by experienced neurologists according to the Parkinson’s Disease Society’s criteria [
An additional 24 age- and gender-matched healthy subjects with no medical history of neurological diseases, brain trauma, psychiatric illnesses, or substance abuse were also enrolled in the control group for comparison. The Local Ethics Committee on Human Research of Kaohsiung Chang Gung Memorial Hospital in Taiwan approves the protocols of this study.
All patients received the Wechsler Adult Intelligence Scale-III (WAIS-III) as the neuropsychiatric survey, conducted by a clinical psychologist blinded to the clinical status of each patient, including assessments of attention, executive, speech and language, memory, and visuospatial functions [
The blood sampling in this study was applied using a 10-ml K3-EDTA tube during the period from 10 : 00 AM to 2 : 00 PM. The centrifugation was performed at 1500-2500 g for 15 minutes with a temperature of 15-25°C by using a swing-out (bucket) rotor. The supernatant from the blood tube was moved to Eppendorf tubes by transferring every 0.5 ml plasma (supernatant) into a 1.5 ml Eppendorf tube. All plasma samples were frozen at -80°C within 3 hours after sampling. The methodology is detailed in our previous studies [
For
The MRI scan protocols and data processing are detailed in our previous study [
All demographic data were compared among the groups using the univariate analysis of variance test and reported as the
To detect between-group gray matter volume differences, a general linear model which was implemented in SPM8 was used to compare modulated gray matter segments. The statistics are detailed in our previous study [
Partial correlation analysis was performed to check the relationships among plasma abnormal protein aggregate levels, brain regional volume, and neuropsychiatric subtest results of the PD groups after controlling for age, gender, and education to minimize the potential influences.
To investigate the possible relationships among abnormal protein aggregate levels, brain regional volume, and neuropsychiatric subtest results, a single-level three-variable mediation model was used with applying the PROCESS macro. An accelerated bias-corrected bootstrap test of statistical significance was performed with 5000 bootstrap samples for SPSS [
The demographic data of the participants are shown in Table
Demographic characteristics of PDN patients, PDCD patients, and control subjects.
Normal control | PDN | PDCD | |||
---|---|---|---|---|---|
Total | PDN vs. PDCD | ||||
Number | 24 | 13 | 18 | ||
Gender (M : F) | 6 : 18 | 7 : 6 | 5 : 13 | 0.182 | 0.131 |
Age | 0.489 | 0.302 | |||
Education (years) | 0.003 | 0.012 | |||
UPDRS I | N/A | 0.937 | |||
UPDRS II | N/A | 0.045 | |||
UPDRS III | N/A | 0.149 | |||
UPDRS 176 | N/A | 0.089 | |||
Modified Hoehn and Yahr staging | N/A | 0.379 | |||
Schwab and England (S&E) scale | N/A | 0.859 | |||
Levodopa equivalent daily doses (mg) | N/A | 0.02 | |||
Plasma abnormal protein aggregates levels (pg/mL) | |||||
NfL | <0.001 | 0.048 | |||
T-tau | 0.001 | 0.350 | |||
AB42 | 0.035 | 0.085 | |||
AB40 | <0.001 | 0.083 | |||
Alpha Synuclein | <0.001 | 0.052 | |||
Wechsler Adult Intelligence Scale-III (WAIS-III) | |||||
Picture completion | 0.002 | 0.035 | |||
Vocabulary | <0.001 | 0.005 | |||
Digit symbol-coding | <0.001 | 0.002 | |||
Similarities | <0.001 | 0.007 | |||
Block design | <0.001 | 0.016 | |||
Arithmetics | <0.001 | <0.001 | |||
Matrix reasoning | 0.006 | 0.008 | |||
Digitspan | <0.001 | <0.001 | |||
Information | <0.001 | 0.147 | |||
Picture arrangement | 0.007 | 0.203 | |||
Comprehension | <0.001 | 0.005 | |||
Letter-number sequencing | 0.037 | 0.010 |
Age and gender data were compared by ANOVA test. Misfolding protein biomarkers were compared by analysis of covariance (ANCOVA) after controlling for age and gender. Wechsler Adult Intelligence Scale-IV was compared by analysis of covariance (ANCOVA) after controlling for age, gender, and education. Data are presented as
The plasma abnormal protein aggregates levels of the participants are listed in Table
The plasma abnormal protein aggregate levels (pg/mL) of the participants showed that the two PD patient groups had significantly increased plasma levels of NfL, T-Tau, and
The two PD patient groups demonstrated elevated plasma NfL levels. More specifically, the PDN patients showed a significantly higher NfL level than the normal controls (
The locations and spatial extents of anatomical regions with significant differences in GMV between the PDN and PDCD groups are presented in Table
Regions showing gray matter volume differences between PD patients with normal cognition (PDN) and cognitive decline (PDCD).
MNI atlas coordinates | Voxel size | Gray matter | BA | GMV (mm3) | ||||
---|---|---|---|---|---|---|---|---|
X | Y | Z | PDN | PDCD | ||||
-32 | -12 | -11 | 753 | Putamen, L | — | 4.18 | ||
14 | -7 | -26 | 108 | Hippocampal complex, R | 34 | 3.56 | ||
27 | 12 | 3 | 388 | Putamen, R | — | 3.47 | ||
57 | -12 | 16 | 187 | Superior temporal gyrus, R | 41 | 3.43 | ||
12 | -25 | 43 | 178 | Cingulate gyrus, R | 31 | 3.2 | ||
50 | 12 | -38 | 187 | Middle temporal gyrus, R | 38 | 3.16 |
Abbreviations: MNI: Montreal Neurological Institute; BA: Brodmann area; GMV: gray matter volume; R: right side; L: left side. The statistical criteria were set at joint voxel height uncorrected
The locations and spatial extents of anatomical regions with significant differences in GMV between the PDN and PDCD groups included bilateral putamen, right hippocampal complex, cingulated gyrus, right superior, and middle temporal gyri.
The summary of the correlations with their
Correlations between neurofilament light chain, certain WAIS subtests, and the specific brain regions of the PD patients.
Correlation ( | ||||||
---|---|---|---|---|---|---|
Putamen, L | Hippocampal complex, R | Putamen, R | Superior temporal gyrus, R | Cingulate gyrus, R | Middle temporal gyrus, R | |
(-32, -12, -11) | (14, -7, -26) | (27, 12, 3) | (57, -12, 16) | (12, -25, 43) | (50, 12, -38) | |
Misfolding protein | ||||||
Neurofilament light chain | -0.297 | 0.052 | -0.096 | -0.462 | -0.224 | 0.071 |
WAIS subtests | ||||||
Picture completion | 0.176 | 0.149 | 0.300 | 0.581 | 0.312 | 0.085 |
Vocabulary | 0.402 | 0.517 | 0.363 | 0.382 | 0.433 | 0.340 |
Digit symbol-coding | 0.298 | 0.137 | 0.123 | 0.421 | 0.199 | 0.129 |
Similarities | 0.305 | 0.418 | 0.358 | 0.338 | 0.454 | 0.357 |
Block design | 0.353 | 0.253 | 0.179 | 0.315 | 0.383 | 0.173 |
Arithmetics | 0.452 | 0.429 | 0.273 | 0.367 | 0.308 | 0.325 |
Matrix reasoning | 0.484 | 0.440 | 0.423 | 0.429 | 0.394 | 0.389 |
Digit span | 0.621 | 0.376 | 0.466 | 0.615 | 0.420 | 0.431 |
Comprehension | 0.279 | 0.402 | 0.358 | 0.316 | 0.300 | 0.370 |
Letter-number sequencing | 0.525 | 0.523 | 0.615 | 0.329 | 0.586 | 0.660 |
Correlations between among neurofilament light chain and the specific brain regions of the PD patients were performed by partial correlation after controlling for age, and gender. Correlations between certain WAIS subtests and the specific brain regions of the PD patients were performed by partial correlation after controlling for age, gender, and education.
The plasma NfL level was negatively correlated with the GMV in the right superior temporal gyrus (
The GMV in the left putamen (-32, -12, -11) correlated with the subtest results of Vocabulary, Arithmetics, Matrix reasoning, Digit span, and Letter-number sequencing. The GMV in the right hippocampal complex (14, -7, -26) correlated with the subtest results of Vocabulary, Similarity, Arithmetics, Matrix reasoning, Digit span, Comprehension, and Letter-number sequencing. The GMV in the right putamen (27, 12, 3) correlated with the subtest results of Matrix reasoning, Digit span, and Letter-number sequencing. The GMV in the right superior temporal gyrus (57, -12, 16) correlated with the subtest results of Picture completion, Vocabulary, Digit symbol-coding, Matrix reasoning, and Digit span. The GMV in the right cingulate gyrus (12, -25, 43) correlated with the subtest results of Vocabulary, Similarity, Block design, Matrix reasoning, Digit span, and Letter-number sequencing. The GMV in the right middle temporal gyrus (50, 12, -38) correlated with the subtest results of Matrix reasoning, Digit span, and Letter-number sequencing.
The primary hypothesis of this analysis was to determine whether the effect of plasma abnormal protein aggregates levels, such as plasma NfL level (independent variable) on neuropsychiatric performance (dependent variable) was explainable directly or indirectly by certain affected brain region volumes (mediator) with a significant group main effect. The path model jointly tested three effects of interest, which are required if the certain affected brain region volume links the plasma abnormal protein aggregates level with the neuropsychiatric performance: (a) the effect of the independent variable (plasma NfL level) on the mediator (the certain affected brain region volume) (indirect effect, path a), (b) the effect of the mediator on the dependent variable (the neuropsychiatric performance) by controlling the effect of the certain affected brain region volume (indirect effect, path b), and (c) the mediation effect a
For simplicity, we report a full list of results from the present study that fulfill the three criteria cited previously. Consequently, the mediation statistics were performed only for the plasma NfL level, the right superior temporal gyrus volume, and neuropsychiatric subtest, results that passed the partial correlation. The mediation relationship models for picture completion and digit span were significant (
The statistics for mediation models were performed only for the plasma NfL level, the right superior temporal gyrus volume, and the neuropsychiatric subtest results that pass the partial correlation. The mediation relationship models for the plasma NfL level, the right superior temporal gyrus volume, (a) picture completion, and (b) digit span after controlling for age, gender, and education. The two models verifying the poorer neuropsychiatric performances in picture completion and digit span were caused by the vulnerable brain regions loss damaged by the elevated plasma NfL level via a full mediation effect.
Consistent with previous studies and our hypothesis, the PDCD group exhibited higher plasma NfL levels, decreased regional brain volume, and poorer neuropsychiatric subtest results compared with the PDN group. Most of the correlations between these clinical presentations showed significance. The full mediation models among plasma NfL level (independent variable), vulnerable regional brain volume (mediator), and the psychiatric subtest results of picture completion and digit span (dependent variable) were revealed. These results indicate that the vulnerable regional brain volume may be a full mediator in the relationship between plasma abnormal protein aggregate levels and poor psychiatric subtest results in PD patients.
Oxidative stress could cause the vulnerable proteostasis network to malfunction, while aging could exacerbate the situation [
This study demonstrates the correlation of the right superior temporal gyrus volume with poor picture completion performance in PD patients. Picture completion ability is associated with visuospatial function, while spatial awareness and spatial neglect associated with the superior temporal gyrus have been shown by studying patients with pure spatial neglect and right brain damage [
Beyond these associations demonstrated in this study, the establishment of mediation models could provide further statistical evidence to verify and further elucidate the causal relationship [
First, as a process of oxidative stress [
Second, mounting evidence indicates that CSF or plasma NfL levels correlate with brain volume, white matter integrity, or even white matter hyperintensity lesions [
Third, from a pathophysiological point of view, oxidative stress could contribute to mesial temporal degeneration [
Finally, as a result of passing the Sobel test in the full-mediation model, it has been suggested that vulnerable regional brain volumes may act as a full mediator in the relationship between plasma abnormal protein aggregate levels and poor psychiatric subtest results in PD patients. Therefore, there would be no direct correlation between plasma abnormal protein aggregate levels and poor psychiatric subtest results if the effects of the vulnerable regional brain volumes were removed. The mediation model investigated in this study indicates that elevated plasma NfL levels caused by oxidative stress and neuro-axonal loss could exacerbate multiple regional brain atrophy and further affect cognitive performance.
Although this study presents a considerable body of evidence, the interpretation of these results should be approached with caution. First, oxidative stress is one cause of plasma abnormal protein aggregate formation; however, there are also influences affecting plasma abnormal protein aggregate levels. Further studies with more oxidative stress biomarkers, like NADPH oxidase 2, hydrogen peroxide, and 8-iso-PGF2
This study demonstrates the association between poor neuropsychiatric subtest results, higher plasma abnormal protein aggregate levels, and decreased regional brain volume in PD patients. Our mediation model indicates the possible pathophysiological importance of vulnerable regional brain volumes in the coexistence of plasma abnormal protein aggregates and poor neuropsychiatric subtest performance. This offers valuable insight into the intricate relationship between poor neuropsychiatric subtest performance, elevated plasma abnormal protein aggregate levels, and decreased regional brain volume in PD patients to further clarify the pathogenesis of PD cognitive decline.
The data that support the findings of this study are available on request from the corresponding author, Meng-Hsiang Chen. The data are not publicly available due to their containing information that could compromise the privacy of research participants.
The authors declare no conflict of interest concerning the research related to the manuscript.
We thank Mrs. Yi-Wen Chen and Mrs. Yi-Hui Wu for taking responsibility for the integrity of the data and the accuracy of the data analysis. This work was supported by funds from the National Science Council (CMRPG8K0221 and CORPG8L0191 to Wei-Che Lin), MOST (108-2314-B-182A-017 to Meng-Hsiang Chen), and Chang Gung Memorial Hospital (CORPG8K0221 and CMRPG8E0621 to Meng-Hsiang Chen).