Supplementary Fig.1. Tyrosine hydroxylase (TH) immunoreactivity in substantia
nigra (left two columns) and putamen (right two columns) from age-matched control
(A-D), H&Y stage 1 PD (E-H), H&Y stage 3 PD (I-L), and H&Y stage 5 PD (M-P)
cases. There was no obvious difference for TH immunoreactive (TH-ir) extensity and
intensity in substantia nigra between H&Y stage 1 PD cases (E, F) and age-matched
control (A, B). However, a severe reduction of TH immunoreactivity was observed in
putamen (G, H); relative to age-matched control (C, D). In H&Y stage 3-5 PD cases,
some remaining nigral NM-laden neurons exhibited TH-ir (arrows; J, N), while others
displayed no detectable TH-ir (arrowheads; J, N). TH-ir fibers were not visible in
major putamen, except ventromedial putamen near globus pallidus (arrows; K, O). At
higher magnification, dense fine TH-ir fibers (< 0.25 µm) consisted of the fine mesh
in gray matter of putamen and composed the fine background TH-ir
immunoreactivity were observed in age-matched control (D). TH-ir fine fibers were
dramatically decreased in H&Y stage 1 PD (H) and hardly detected in H&Y stage 3-5
PD (L, P). Morphologically abnormal, thick TH-ir fibers (>0.5 µm) displaying
swollen varicosities and intervaricose segments were observed in all PD cases
(arrows, H, L, P). Scale bar in M = 0.4mm (applies to
80µm (applies to
A, E, I); Scale bar in N =
B, F, J); Scale bar in O = 1.6mm (applies to C, G, K); Scale bar in
P= 16µm (applies to D, L, H, P).
Supplementary Fig. 2. Confocal microscopic images of substantia nigra from agematched control (A1-3), H&Y stage 2 PD (B1-3), and H&Y stage 5 PD (C1-3)
showing immunostaining patterns for kinesin light chain 1 (KLC1; green; A1, B1,
C1), tyrosine hydroxylase (TH; red; A2, B2, C2) and co-localization of KLC1 and TH
(Merged; A3, B3, C3). Note that in H&Y stage 2 PD, KLC1 immunofluorescence
intensity was markedly reduced in neurons (arrows; B1, B3) displaying intensive TH
staining (B2) comparable to that observed in neurons of age-matched control cases
(A2). In H&Y stage 5 PD, both TH ad KLC1 immunoreactivities were dramatically
reduced in remaining NM-laden nigral neurons. Scale bar in C3= 160 µm (applies to
all).
Supplementary Fig. 3. Confocal microscopic images of substantia nigra from agematched control (A-C) and H&Y stage 2 PD (D-F) illustrating immunostaining for
dynein light chain Tctex type 3 (DYNLT3; green; A, D), alpha-synuclein (-syn; red;
B, E) and co-localization of DYNLT3 and -syn (Merged; C, F). Note that DYNLT3
immunofluorescence intensity was reduced in nigral neurons with -syn inclusions
(arrows D-F) in PD. Some nigral neurons without -syn inclusion (arrowheads; D, E)
in PD exhibited intensive DYNLT3 labeling similar to age-matched control. Scale
bar in F= 120µm (applies to all).
Supplementary Fig. 4. Photomicrographs of nigral (A-F) and striatal (G-L) sections
obtained from rats injected with adeno-associated viruses encoding either mutant
A30P human -synuclein (AAV-A30P; A-D; G-J), or green fluorescent protein
(AAV-GFP; E, F, K, L). Immunohistochemistry illustrates the patterns of -synuclein
(-syn; A, B, G, H) and tyrosine hydroxylase (TH; C-F; I-L) immunoreactivity. Note
the pronounced expression of -syn in nigrostriatal system (A, B, G, H) and the
accumulation of -syn in nigral neurons (arrows; A, B) and striatal fibers (arrows; G,
H) of rats injected with adenoviruses encoding mutant A30P human -syn. Axonal
fibers filled with -syn displayed swelling of intervaricose segments (arrows; H) - a
hallmark of axonal injury. Target expression of A30P human -syn resulted in a
reduction of TH levels in the nigrostriatal system (arrows; C, D, I) and the swelling of
axons (arrows; J) in the striatum. In contrast, robust TH-ir was observed in soma (E,
F) and processes (K, L) of nigrostriatal neurons expressing GFP. Scale bar 90µm in D
(applies to B, F); 1.8 mm for A, C, E; 1.2mm for G, I, K; 30µm for H, J, L.
Supplementary Fig. 5. Confocal microscopic images of substantia nigra from
uninjected rats illustrating immunostaining for kinesin heavy chain (KHC; green; A),
dynein light chain Tctex type 3 (DYNLT3; green; D), tyrosine hydroxylase (TH; red;
B, E) and co-localization of TH and KHC (Merged; C) or DYNLT3 (Merged; F).
Note that all TH-ir neurons were KHC and DYNLT3 immunopositive. Some non-THir neurons also expressed these markers (arrows; C, F). Scale bar in F= 140µm
(applies to all).
Supplementary Fig. 6. Laser confocal microscopic images of substantia nigra
illustrating human -synuclein (-syn; green; A), dynein light chain Tctex type 3
(DYNLT3; red; B), and the merged (C) from rats with targeted expression of human
mutant (A30P) -syn and green fluorescent protein (GFP; green; D), DYNLT3 (red;
E), and merged (F) from rat with target expression of GFP. Note that DYNLT3-ir was
not detected in neurons displaying strong -syn-ir (A-C; arrows), but was preserved
in neurons without -syn-ir (B, C; arrowheads) cells. In contrast, there was no
obvious difference in DYNLT3-ir intensity between GFP-positive (D-F; arrows) and
negative (E, F; arrowhead) nigral neurons. Scale bar in F=120 µm (applies to all).