Sacino et al Supplemental Information 1
Supplementary Material
Title: Non-prion type transmission in A53T α-synuclein transgenic mice: A normal
component of spinal homogenates from naïve non-transgenic mice induces robust αsynuclein pathology
Journal name: Acta Neuropathological
Authors: Amanda N. Sacino, Jacob I. Ayers, Mieu Brooks, Paramita Chakrabarty, Vincent J.
Hudson III, Jasie K. Howard, Todd E. Golde, Benoit I. Giasson and David R. Borchelt
Corresponding Author: Address correspondence to Benoit I. Giasson (bgiasson@ufl.edu) or
David R. Borchelt (drb1@ ufl.edu)
Inventory of Supplementary Material:
1. Materials and Methods
2. Supplementary Bibliography
3. Supplemental Figures 1, 2 and 3
Material and Methods
Mouse husbandry and stereotactic injections: All procedures were performed according to the
NIH Guide for the Care and Use of Experimental Animals and were approved by the University
of Florida Institutional Animal Care. M83 transgenic mice expressing human A53T αS with
expression driven by the mouse prion protein promoter [4] and G93A hSOD1 in B6SJLTgN(SOD1-G93A)1Gur mice were described previously [5]. These lines along with the
wildtype, non-transgenic (NTg) mice, were maintained in a hybrid background of C57BL/6 and
C3H/HeJ. αS null mice previously described [1] were obtained from The Jackson Laboratory
Sacino et al Supplemental Information 2
(Bar Harbor, MA). For stereotactic intracerebral injections of brain homogenates, all mice at 2-3
months of age were bilaterally stereotaxically injected with the homogenates listed in Table 1 in
the hippocampus (coordinates from Bregma: A/P -1.7, L +/-1.6, D/V -2.0). The inoculum was
injected at a rate of 0.2 ul per min with the needle in place for 15 min at each site. The CNS
homogenates summarized in Table 1 were prepared by lysing the tissue in sterile PBS with two
consecutive bursts of 3-seconds with a hand held metal probe sonicator (Misonix microson
XL2000 ultrasonic cell disruptor) followed by sedimentation at 1000 g for 5 minutes. The metal
probe sonicator was extensively cleaned between applications. Protein concentration was
determined with the bicinchoninic acid (BCA) assay (ThermoFisher Scientific, Grand Island,
NY) using bovine serum albumin as the standard.
Antibodies: pSer129, also known as clone 81A, is a mouse monoclonal antibody that reacts with
αS phosphorylated at Ser129 [8]. The rabbit monoclonal antibody pSer129/EP1536 that reacts
with αS phosphorylated at Ser129 and does not cross-react with phosphorylated low-molecularmass neurofilament subunit [6] was obtained from Abcam (Cambridge, MA). Syn506 is
conformational anti-αS mouse monoclonal antibody that preferentially detects αS in pathological
inclusions [3, 7]. A rabbit anti-p62/sequestosome polyclonal antibody (SQSTM1) was obtained
from Proteintech (Chicago, IL). Neuronal specific mouse monoclonal anti-III tubulin antibody
TuJ-1 was purchased from Fisher (Hanover Park, IL). Anti-vimentin (C20) rabbit antibody was
from Santa Cruz Biotechnology Inc (Dallas, TX). Anti-glial fibrillary acidic protein (GFAP)
mouse monoclonal antibody GA5 was obtained from Cell Signaling Technology (Danvers, MA).
Immunohistochemical analysis: Mice were sacrificed with CO2 euthanization and perfused with
PBS/heparin. The brain and spinal cord (SC) were then removed and fixed with 70%
ethanol/150mM NaCl for at least 48 hours. As previously described, tissues were dehydrated at
Sacino et al Supplemental Information 3
room temperature through a series of ethanol solutions, followed by xylene and then were
infiltrated with paraffin at 60C [2]. The tissues were then embedded into paraffin blocks, which
were cut into 7 μm sections. Immunostaining of the sections was performed using previously
described methods [2] using avidin-biotin complex (ABC) system (Vectastain ABC Elite Kit,
Vector Laboratories, Burlingame, CA) and immunocomplexes were visualized with the
chromogen 3,3’-diaminobenzidine. Sections were counterstained with hematoxylin. All slides
were scanned using an Aperio ScanScope CS (40× magnification; Aperio Technologies Inc.,
Vista, CA) and images of representative areas of S pathology were taken using the
ImageScopeTM software (40× magnification; Aperio Technologies Inc.).
Double-labeling immunofluorescence analysis: Paraffin-embedded tissue sections were
deparaffinized and hydrated through a series of graded ethanol solutions followed by 0.1M Tris,
pH 7.6. The sections were blocked with 5% dry milk/0.1M Tris, pH 7.6 and were incubated
simultaneously with combinations of primary antibodies diluted in 5% dry milk/0.1M Tris, pH
7.6. After extensive washing, sections were incubated with secondary antibodies conjugated to
Alexa 594 or Alexa 488 (Invitrogen, Eugene, OR). Sections were post-fixed with formalin,
incubated with Sudan Black, and stained with 4',6-diamidino-2-phenylindole (DAPI)(Invitrogen,
Eugene, OR). The sections were coverslipped with Fluoromount-G (SouthernBiotech,
Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71
Olympus digital camera to capture images.
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References
1
Abeliovich A, Schmitz Y, Farinas I, Choi-Lundberg D, Ho WH, Castillo PE, Shinsky N,
Verdugo JM, Armanini M, Ryan Aet al (2000) Mice lacking alpha-synuclein display
functional deficits in the nigrostriatal dopamine system. Neuron 25: 239-252
2
Duda JE, Giasson BI, Gur TL, Montine TJ, Robertson D, Biaggioni I, Hurtig HI, Stern
MB, Gollomp SM, Grossman Met al (2000) Immunohistochemical and biochemical
studies demonstrate a distinct profile of alpha-synuclein permutations in multiple system
atrophy. J Neuropathol Exp Neurol 59: 830-841
3
Duda JE, Giasson BI, Mabon ME, Lee VM-Y, Trojanoswki JQ (2002) Novel antibodies
to oxidized -synuclein reveal abundant neuritic pathology in Lewy body disease. Ann
Neurol 52: 205-210
4
Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM (2002) Neuronal
alpha-synucleinopathy with severe movement disorder in mice expressing A53T human
alpha-synuclein. Neuron 34: 521-533 Doi S0896627302006827 [pii]
5
Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, Caliendo J,
Hentati A, Kwon YW, Deng HXet al (1994) Motor neuron degeneration in mice that
express a human Cu,Zn superoxide dismutase mutation. Science 264: 1772-1775
6
Uchihara T, Giasson BI (2015) Propagation of alpha-synuclein pathology: hypotheses,
discoveries, and yet unresolved questions from experimental and human brain studies.
Acta Neuropathol: Doi 10.1007/s00401-015-1485-1
7
Waxman EA, Duda JE, Giasson BI (2008) Characterization of antibodies that selectively
detect alpha-synuclein in pathological inclusions. Acta Neuropathol 116: 37-46
Sacino et al Supplemental Information 5
8
Waxman EA, Giasson BI (2008) Specificity and regulation of casein kinase-mediated
phosphorylation of alpha-synuclein. J Neuropathol Exp Neurol 67: 402-416
Figure Legends
Supplement Figure 1. Induction of S inclusion pathology in M83+/- mice following
intracerebral injections of SC homogenates from aged, motor impaired M83+/+ mice.
Representative images showing S inclusion pathology in the hippocampus (a) and entorhinal
cortex (b) of M83+/- mice following the hippocampal injection of SC homogenates from motor
impaired M83+/+ mice. Sections were staining with antibodies pSer129/EP1536Y, pSer129/81A,
Syn 506 or anti-p62/sequestosome as indicated in each panel. Tissue sections were
counterstained with hematoxylin. Scale bar = 100m
Supplemental Figure 2. S inclusion pathology in glial and neuronal cells of M83+/- mice
following intracerebral injections of mouse SC homogenates. Representative double-immuno
fluorescence images showing S inclusion pathology in the hippocampus (a, b) or cortex (c, d)
of M83+/- mice following the hippocampal injection of SC homogenates from motor impaired
M83+/+mice. As indicated tissue sections were double labeled with antibodies to (a) pSer129/S81A and vimentin, (b) GFAP and pSer129/S-EP1536Y, (c) GFAP and pSer129/S-EP1536 or
(d) neuronal specific -tubulin-TuJ-1 and pSer129/S-EP1536Y. Sections were also staining
with DAPI (blue) and the overlays are shown in the images on the right. Arrows depict S
inclusions present in glial cells. Scale bar = 50 m.
Sacino et al Supplemental Information 6
Supplemental Figure 3. Induction of S inclusion pathology in M83+/- mice following
intracerebral injections of SC homogenates from NTg and S null mice, but not from the
cortical brain homogenate from NTg mice. Representative images showing S inclusion
pathology in the hypothalamus, periaqueductual gray, pons and spinal cord regions of M83+/mice following hippocampal injection of SC homogenates from NTg mice 105 days post
injection (NTg-SC*) and 180 days post injection (NTg-SC) or of SC homogenates from S null
mice 135 days post injection (S null-SC). No S inclusion pathology was observed in M83+/mice following hippocampal injection of cortical brain homogenates from NTg mice 135 days
post injection (NTg-CX). Sections were staining with antibodies pSer129/EP1536Y. Tissue
sections were counterstained with hematoxylin. Scale bar = 100m