Brain samples and neuropathology
The present study included eight PD patients and five control subjects. Post-mortem brain samples from neuropathologically-confirmed cases of PD brain samples were obtained from the National Hospital Organization Hyogo-Chuo Hospital (Sanda, Hyogo, Japan), Kobe City Medical Center General Hospital (Kobe, Hyogo, Japan), and Kobe University Hospital (Kobe, Hyogo, Japan). We used five autopsy brains of subjects without parkinsonism and Lewy pathology from the National Hospital Organization Hyogo-Chuo Hospital as control. We reviewed medical records and collected clinical information of each patient. Written informed consent was obtained from the next of kin. This study was approved by the ethical committee of Kobe University Hospital.
We examined brain samples as previously reported in accordance with protocols in Research Resource Network Japan [38, 42]. Briefly, we stored portions of brain tissues were stored at − 80 °C and the rest of brain was fixed in 10% neutral buffered formalin. After fixation, the cerebrum was serially sliced in a coronal plane, the brainstem in an axial plane, and the cerebellum in a sagittal plane. Representative anatomical regions were embedded in paraffin. Serial 6-µm-thick sections were stained with hematoxylin and eosin (H&E). The following regions were examined: dorsal motor nucleus of the vagus, medullary raphe nuclei, locus coeruleus, substantia nigra, raphe nuclei of the midbrain, amygdala, entorhinal cortex, hippocampus, putamen, caudate, and neocortex (frontal, temporal, parietal, and occipital). Two continuous sections of each region were analyzed: one with phosphorylated-αSYN immunohistochemistry and one with αSYN-PLA.
Clinical information and diagnosis of patients
All patients included in the present study were examined by multiple board-certificated neurologists between 2006 and 2017. We reviewed the medical records of each patient and collected the following clinical information: sex, age at onset, age at death, initial symptoms, visual hallucinations, and cognitive impairment. We considered a patient to have visual hallucinations if there were documented complaints from the patient or family members. A patient was considered to have cognitive impairment if a physician had diagnosed cognitive abnormalities, such as memory disturbance, disorientation, executive dysfunction, or stereotypic behavior [15]. We confirmed the pathological diagnosis of PD based on the presence of moderate to severe neuronal loss in the substantia nigra and the presence of LRP [14], which was assessed with H&E-stained sections and phosphorylated-αSYN immunohistochemistry of the midbrain. Control subjects were processed the same and had no neuronal loss or LRP in the substantia nigra.
Immunohistochemistry
Immunohistochemistry for phosphorylated-αSYN was performed as described previously [38]. Paraffin-embedded brain sections were dewaxed in xylene, then rehydrated in a graded series of alcohol. Antigen retrieval was performed by microwave heating of slides for 15 min in pH6 citrate buffer. After blocking in 3% bovine serum albumin in phosphate-buffered saline at room temperature for 30 min, primary antibody for phosphorylated-αSYN (1:2000; mouse monoclonal, psyn#64, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added and incubated at 4 °C overnight. Sections were washed in Tris-buffered saline (TBS) and were then incubated in hydrogen peroxide at room temperature for 30 min to inactivate endogenous peroxidase activity. After washing the sections, they were incubated with a biotin-conjugated secondary antibody (goat anti-mouse IgG) and then with avidin–biotin complex (VECTASTAIN Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA). Phosphorylated-αSYN was visualized with 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate and the sections were counterstained with hematoxylin. Samples were dehydrated in a graded series of alcohol and xylene before mounting with Permount mounting medium (Falma, Tokyo, Japan).
Braak neurofibrillary tangle (NFT) stage [7] and Thal amyloid phase [41] were assigned by thioflavin S fluorescent microscopy as previously described [37]. Immunohistochemistry for tau (AT8, mouse monoclonal, 1:2500, Thermo Fisher Scientific, Rockford, IL, USA) and phosphorylated transactive response DNA-binding protein of 43 kDa (pTDP-43) (pS409/410, mouse monoclonal, 1:5000, Cosmo Bio USA, Carlsbad, CA, USA) were performed on the hippocampus section of PD patients as previously described [31].
αSYN-PLA staining
We conducted αSYN-PLA staining by using Duolink kits supplied by Sigma-Aldrich (St. Louis, MO, USA) to detect αSYN oligomers following the manufacturer instructions. We made both PLA probes with an αSYN antibody (mouse monoclonal, Syn211, Abcam, Cambridge, UK). We added 20 µg of Syn211 antibody to 2 µl of conjugation buffer and transferred the solution to a vial containing lyophilized oligonucleotides (plus or minus). Then, we incubated the solution at room temperature overnight. The conjugates were incubated with 2 µl of stop solution for 30 min at room temperature and suspended in 24 µl of storage solution. After dewaxing and hydrating the tissue sections as above, sections were incubated with hydrogen peroxide for 1 h at room temperature and subsequently heated in a microwave for 15 min in pH6 citrate buffer. Sections were blocked in 3% bovine serum albumin in phosphate-buffered saline at 37 °C for 1 h, followed by incubation with PLA probes diluted in PLA probe diluent (1:100) at 37 °C for 1 h, and then at 4 °C overnight. Sections were washed in TBS-T (TBS + 0.05% Tween 20) and then incubated with ligation solution and ligase at 37 °C for 1 h, washed in TBS-T and then incubated with amplification reagents and polymerase at 37 °C for 2.5 h. Finally, the sections were washed in TBS-T, incubated with detection solution at room temperature for 1 h, and finally incubated with substrate solution at room temperature for 20 min. Sections were counter-stained with hematoxylin and dehydrated in a graded series of alcohol and xylene before mounting with a bright-field mounting medium.
Evaluation of LRP and αSYN oligomer burden
We assessed LRP (Lewy bodies and Lewy neurites) on phosphorylated-αSYN immunostained slides using a semi-quantitative scoring scheme at 20 × magnification. The severity of LRP was rated on a five-point scale: absent (0), slight (1+), mild (2+), moderate (3+), and severe (4+). A Braak PD stage was assigned to each case based on the distribution of LRP [8, 9].
We evaluated αSYN oligomer burden on αSYN-PLA slides as previously described [38]. Each stained slide was viewed at 20 × magnification and evaluated for the pattern and severity of neuronal and neuropil staining. Neuronal staining was classified based upon the staining pattern as follows: neuronal-clustered, neuronal-patchy, neuronal-punctate, and null (no signal detected) (Fig. 1A). Neuronal-clustered was characterized by αSYN-PLA signal throughout the neuronal perikarya; neuronal-patchy had patchy αSYN-PLA signal in the neurons; neuronal-punctate had dot-like αSYN-PLA signal. Neuropil staining was rated on a scale from zero (no signal) to five (highest signal) by using pre-made scoring plates (Fig. 1B). Because the cortical areas were large, we eliminated bias by evaluating αSYN-PLA neuropil scores in the three most affected microscopic fields and calculating the average scores in the cortex of superior frontal gyrus, superior temporal gyrus, superior parietal lobule, lingual gyrus, and parahippocampal gyrus. In other regions, such as the dorsal motor nucleus of the vagus, medullar raphe nuclei, locus coeruleus, substantia nigra, raphe nuclei of the midbrain, amygdala, hippocampus, putamen, and caudate, the region with the most signal was scored.
To compare the severity of αSYN oligomer burden with LRP, we made a combined severity score for αSYN oligomers on a five-point scale from the neuronal staining pattern and neuropil scores. We compared the score of LRP severity and combined severity of αSYN oligomer burden in each brain region. We also compared the mean pathology scores of each patient in the brainstem and neocortex to compare regional anatomical differences in the distribution of each pathology.
In addition to the scoring described above, we quantitatively measured the immunoreactive area of αSYN-PLA staining using the software ImageJ (National Institute of Health, Bethesda, MD, USA). The cortical images were used because they were large enough to fill all areas of 1720 × 1075 pixels at 20 × magnification. Three representative images of each cortex were obtained with the Aperio AT2 Slide Scanner (Leica Biosystems, Deer Park, IL, USA) at 20 × magnification. Quantification of immunostaining was performed using the IHC Image Analysis Toolbox plugin. After the training procedure with Nova Red color, the stained images were analyzed with color detection and converted to a 16-bit format (Additional file 1: Fig. S1A). Then, we measured the area of threshold pixels.
Statistical analysis
We conducted all statistical analysis with GraphPad Prism (version 9.1.2, GraphPad Software, La Jolla, CA, USA). Unpaired t test was used to compare differences in age at death between PD and controls. We compared the LRP scores and combined αSYN oligomer score in each brain region by Wilcoxon matched-pairs signed-rank test. The combined αSYN oligomer scores and pathological burden scores in each brain region of PD and controls were compared with respect to clinical symptoms with unpaired t test (Welch’s correction was performed when the variance of the two groups was not equal). We calculated Spearman's rank correlation coefficient and examined whether there is a correlation between the duration of disease and the respective pathology scores. Statistical significance was defined by a p value < 0.05.