Pathological α-synuclein Distribution in Subjects with Coincident Alzheimer’s and Lewy Body Pathology

Jon B. Toledo, PallaviGopal, Kevin Raible, David J. Irwin, Johannes Brettschneider, Samantha Sedor, Kayla Watts, Susana Boluda, Murray Grossman, Vivianna M. Van Deerlin, Edward B. Lee, Steven E. Arnold, John E. Duda, Howard Hurtig, Virginia M-Y Lee,CharlesH. Adler, Thomas G. Beach, John Q. Trojanowski

BBDP Olfactory bulb and extra-cranial tissue

As detailed previously[1,3,2], olfactory bulbs and tracts were removed from the cerebrum and embedded intact to give longitudinal sections. Approximately 2 cm of vagus nerve was obtained from alongside the common carotid artery at the level of the larynx, with approximately one-third of this cut into cross-sections (2-3 cross-sections) while the remainder was embedded intact to give longitudinal sections. Submandibular gland samples, approximately 1.5 x 1 x 0.4 cm, were cross-sections through the middle of the gland. Lower esophagus samples were taken at a level 2-3 cm proximal to the gastro-esophageal junction and consisted of the full-thickness of the esophageal wall, cut into strips measuring approximately 2 x 0.4 x 0.3 cm, with two strips embedded per cassette. Spinal cord cross-sections, approximately 0.4 cm thick, were taken from approximate levels of the exit of cervical nerve roots 4-6, thoracic nerve roots 5-7 and lumbar nerve roots 4-5. Two cross-sections were embedded into one cassette. For this study, only data from lumbar spinal cord levels were used. As for brain tissue, spinal cord and peripheral tissue samples were fixed for 2 days at 4°C in a commercial formalin preparation containing 4% formaldehyde and thereafter dehydrated and embedded in paraffin wax. All of the olfactory bulb, spinal cord, submandibular gland and lower esophagus samples were contained within a single cassette and between 1 and 3 sections, at 6 µm, were obtained from each, mounted on separate slides and stained for LRP. The IHC method used was identical to that used for the brain studies. For some samples of submandibular gland and esophagus, during initial exploratory studies, additional paraffin sections and/or 40 µm sections were obtained on a sledge-type freezing microtome and stained for LRP. After this exploratory phase, single sections of olfactory bulb and spinal cord were stained for LRP (containing one section of olfactory bulb and two cross-sections of spinal cord), while for vagus nerve, submandibular gland and esophagus, 3 sections from each were regularly stained for LRP (each section containing one cross-section of submandibular gland and two strips of esophagus).

Supplementary Table 1. Areas stained and graded for α-synuclein in each of the two centers.

Center / OB / Medulla / Pons / Midbrain / Thalamus / LN / Amygdala / CA1/ Subiculum / Entorhinal cortex / ACG / SMT / MFG / IPL / Occ
CNDR / Yes / Medulla / Pons
LC / Midbrain
SN / Yes / Putamen
GP / Yes / Yes / Yes / Yes / Yes / Yes / Yes / Yes
BBDP / Yes / DMNV / LC / SN / Yes / Yes / Yes / Yes / Yes / Yes

ACG: Anterior cingulate gyrus; CA: Cornus Ammonis;DMNV: Dorsal motor nucleus of the vagus nerve; GP: Globus pallidus;IPL: Inferior parietal lobe; LC: Locus coeruleus;LN: Lentiform nucleus; MFG: Middle frontal gyrus; Occ: Occipital lobe including primary visual area; SMT: Superior and middle temporal gyrii.

Supplementary Table 2. Staining techniques for each of the deposits included in the study.

Center / Amyloid / α-Synuclein / Tau
CNDR / Thioflavine S / Syn3031[4] / PHF-1 [6]
BBDP / Thioflavine S / pS129[5] / Gallyas

1During one and a half years 81A antibody was used instead of Syn303.

Supplementary Table 3.CERAD score and NFTBraak stages for the different clusters.

CNDR Cohort
Diagnosis / Cluster 1 / Cluster 2 / Cluster 3 / Cluster 4 / Cluster 2A / Cluster 2B / Cluster 2C
CERAD score / 0: 37.0%
A: 9.3%
B: 9.3%
C: 44.4% / 0: 23.3%
A: 5.4%
B: 16.3%
C: 55.0% / 0: 0%
A: 0%
B: 3.8%
C: 96.2% / 0: 0%
A: 0%
B: 9.4%
C: 90.6% / 0: 45.2%
A: 16.1%
B: 19.4%
C: 19.4% / 0: 12.3%
A: 0%
B: 12.3%
C: 75.4% / 0: 22.5%
A: 5.0%
B: 20.0%
C: 52.5%
NFTBraak Stage / 0: 9.3%
I-II: 40.7%
III-IV: 9.3%
V-VI: 40.7% / 0: 2.3%
I-II: 26.9%
III-IV: 27.7%
V-VI: 43.1% / 0: 0%
I-II: 0%
III-IV:3.80%
V-VI: 96.2% / 0: 0%
I-II: 0%
III-IV: 0%
V-VI: 100% / 0: 3.2%
I-II: 58.1%
III-IV: 22.6%
V-VI: 16.1% / 0: 1.7%
I-II: 10.3%
III-IV: 22.4%
V-VI: 65.5% / 0: 2.5%
I-II: 27.5%
III-IV: 40.0%
V-VI: 30.0%
BBDP Cohort
Diagnosis / Cluster 1 / Cluster 2 / Cluster 3 / Cluster 4 / Cluster 5
CERAD score / 0: 9.6%
A: 5.6%
B: 21.2%
C: 63.5% / 0: 27.5%
A: 14.3%
B: 28.6%
C: 29.7% / 0: 0%
A: 0%
B: 12.1%
C: 87.9% / 0: 0%
A: 0%
B: 18.2%
C: 81.8% / 0: 35.3%
A: 8.8%
B: 11.8%
C: 44.1%
NFTBraak Stage / 0: 0%
I-II: 9.6%
III-IV: 50.0%
V-VI: 40.4% / 0: 0%
I-II: 24.2%
III-IV: 56.1%
V-VI: 19.7% / 0: 0%
I-II: 0%
III-IV: 9.1%
V-VI: 90.9% / 0: 0%
I-II: 0%
III-IV: 9.0%
V-VI: 91.0% / 0: 0%
I-II: 32.3%
III-IV: 23.6%
V-VI: 44.1%

Supplementary Table 4.Clinical diagnoses by cluster in the different neuropathological groups in the CNDR cohort.

Dem-AD-LB
Diagnosis / Cluster 1 / Cluster 2 / Cluster 3 / Cluster 4 / Cluster 2A / Cluster 2B / Cluster 2C
AD / 80.0% / 51.0% / 66.0% / 68.8% / 60.0% / 55.5% / 22.2%
DLB/CBS / 10.0% / 31.4% / 15.1% / 9.4% / 0.0% / 30.6% / 55.6%
Other / 10.0% / 17.6% / 18.9% / 21.9% / 40.0% / 13.9% / 22.2%
PD/PD-AD
Diagnosis / Cluster 1 / Cluster 2 / Cluster 3 / Cluster 4 / Cluster 2A / Cluster 2B / Cluster 2C
PD no dementia / 58.8% / 21.7% / - / - / 38.1% / 5.3% / 20.7%
PDD / 41.2% / 78.3% / - / - / 61.9% / 94.7% / 79.3%

Supplementary Table 5.Correspondence of clusters across the BBDP and CNDR cohorts.

CNDR cohort / BBDP cohort
Olfactory bulb cluster / - / 4
Amygdala cluster / 3 / -
Limbic predominant cluster / 4 / 3
Limbic transitional cluster / 1 / 5
Low burden neocortical cluster / 2A / 2
Moderate burden neocortical cluster / 2C / 1
Occipital neocortical cluster / 2B / -

Supplementary Figure 1.Example of area selected for counting of substantia nigra cell density.

Supplementary Figure 2. Evaluation of clustering solutions for first CNDR cluster analysis

Supplementary Figure 3. Evaluation of clustering solutions for CNDRsubclustering analysis

Supplementary Figure 4. Evaluation of clustering solutions for BBDP clustering analysis

Supplementary Figure 5.Representative images of putamen DAT IHC in the different clinico-pathological groups and clusters.


References

1. Beach TG, Adler CH, Sue LI, Serrano G, Shill HA, Walker DG, Lue L, Roher AE, Dugger BN, Maarouf C, Birdsill AC, Intorcia A, Saxon-Labelle M, Pullen J, Scroggins A, Filon J, Scott S, Hoffman B, Garcia A, Caviness JN, Hentz JG, Driver-Dunckley E, Jacobson SA, Davis KJ, Belden CM, Long KE, Malek-Ahmadi M, Powell JJ, Gale LD, Nicholson LR, Caselli RJ, Woodruff BK, Rapscak SZ, Ahern GL, Shi J, Burke AD, Reiman EM, Sabbagh MN (2015) Arizona Study of Aging and Neurodegenerative Disorders and Brain and Body Donation Program. Neuropathology : official journal of the Japanese Society of Neuropathology 35 (4):354-389. doi:10.1111/neup.12189

2. Beach TG, Adler CH, Sue LI, Vedders L, Lue L, White Iii CL, Akiyama H, Caviness JN, Shill HA, Sabbagh MN, Walker DG (2010) Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders. Acta neuropathologica 119 (6):689-702. doi:10.1007/s00401-010-0664-3

3. Beach TG, Sue LI, Walker DG, Roher AE, Lue L, Vedders L, Connor DJ, Sabbagh MN, Rogers J (2008) The Sun Health Research Institute Brain Donation Program: description and experience, 1987-2007. Cell and tissue banking 9 (3):229-245. doi:10.1007/s10561-008-9067-2

4. Duda JE, Giasson BI, Mabon ME, Lee VM, Trojanowski JQ (2002) Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases. Annals of neurology 52 (2):205-210. doi:10.1002/ana.10279

5. Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio K, Iwatsubo T (2002) alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nature cell biology 4 (2):160-164. doi:10.1038/ncb748

6. Greenberg SG, Davies P (1990) A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America 87 (15):5827-5831

1