www.sciencesignaling.org/cgi/content/full/2/80/ra36/DC1

Supplementary Materials forInhibition of mTOR Signaling in Parkinson’s Disease Prevents LDOPA–Induced Dyskinesia
Emanuela Santini, Myriam Heiman, Paul Greengard, Emmanuel Valjent, Gilberto
Fisone**To whom correspondence should be addressed. E-mail: gilberto.fisone@ki.se
Published 21 July 2009, Sci. Signal. 2, ra36 (2009)
DOI: 10.1126/scisignal.2000308

This PDF file includes:
Fig. S1. Rapamycin does not affect L-DOPA–induced phosphorylation of GluR1 andERK2.
Fig. S2. Rapamycin does not affect L-DOPA–induced phosphorylation of Lys14acetylated histone H3.
Fig. S3. Evaluation of the degree of DA denervation after striatal 6-OHDA injection.
Table S1. Effect of 6-OHDA and L-DOPA on selected proteins involved in mTOR
signaling.

Fig. S1. Rapamycin does not affect L-DOPA-induced phosphorylation of GluR1 andERK2. Representative Western blots (top) and quantification (bottom).

Error bars

represent SEM (n = 8-16 mice per treatment) of data calculated as percent of Shamtreated with L-DOPA. Statistical significance was determined by one-way ANOVA
followed by the Bonferroni-Dunn test. *** p < 0.001 versus sham treated with L-DOPA.

Fig. S2. Rapamycin does not affect L-DOPA-induced phosphorylation of Lys14acetylated histone H3. Immunofluorescent detection (A) and quantification (B) of cells
positive for phospho-Ser10-acetyl-Lys14-H3 in the striata of sham- and 6-OHDA-lesioned
mice treated with L-DOPA alone, or in combination with Rapamycin (Rap). Scale bar: 40
m. Error bars represent SEM (n = 3 mice per treatment). Statistical significance wasdetermined by two-way ANOVA followed by the Bonferroni-Dunn test. *** p < 0.001
versus sham treated with L-DOPA.

Fig. S3. Evaluation of the degree of DA denervation following striatal 6-OHDA
injection. Upper panels show lack of tyrosine hydroxylase (TH)-positive fibers in thedorsal striatum (DStr) of a mouse with unilateral lesion of nigrostriatal dopamine system(left) compared with a sham-lesioned mouse (right). Scale bar: 300 m. Lower panels,
show the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc)
following striatal 6-OHDA lesion. Scale bar: 200 m.

Sham

6-OHDA/
Acute DOPA

High Dys(AIMs≥35)

Low Dys(AIMs≤20)

P-Thr389-S6K

100±24220±21 *

248±11 *165±9 †°

421/424100±4150±9 *

151±6 *

110±1 †°

P-Thr

-S6K

P-Ser

240/244-S6100±7224±28 *

270±22 *126±6 †°

P-Ser

235/236-S6

100±4281±18 *

326±22 *138±7 †°-Mnks

100±3162±8 *

163±5 *

116±3 †°

-eIF4E

100±7170±15 *

160±9 *

112±2 †°

P-Ser -4E-BP

100±7148±13 *

164±9 *

106±2 †°

197/202

P-ThrP-Ser

209
65

Table S1. Effect of 6-OHDA and L-DOPA on selected proteins involved in mTORsignaling. Determination of protein phosphorylation in the striata of sham-lesioned mice(Sham), 6-OHDA-lesioned mice treated acutely with L-DOPA (6-OHDA/Acute DOPA)
and 6-OHDA-lesioned mice treated for 10 days with L-DOPA and displaying high (HighDys) or low dyskinesia (Low Dys). High Dys and Low Dys mice were selected based ona total AIMs score ≥35 and ≤20, respectively. Data are calculated as means ± SEM (n =6-18 mice per treatment). Statistical significance was determined by one-way ANOVA
followed by the Bonferroni-Dunn test. * p < 0.001 versus Sham group; † p<0.001 versus
6-OHDA/Acute DOPA group; and ° p<0.001 versus High Dys group.