NICOTINE PREVENTS SYNAPTIC IMPAIRMENT INDUCED BY AMYLOID-

OLIGOMERS THROUGH 7-NICOTINIC ACETYLCHOLINE RECEPTOR

ACTIVATION

Nibaldo C. Inestrosa, Juan A. Godoy, Jessica Y. Vargas, Macarena S. Arrazola,

Juvenal A. Rios, Francisco J. Carvajal, Felipe G. Serrano and Ginny G. Farias.

Supplementary figures

Supplementary figure S1: Nicotine treatment stabilizes and rescues PSD-95 from damage by A oligomers in cultured hippocampal neurons. Western blot for PSD-95 protein in hippocampal neurons challenged with A oligomers (5 M) with or without stimulation with nicotine (10M) or -Btx (5 nM) (A). Western blot for PSD-95 protein from hippocampal neurons challenged wit A oligomers plus Wortmanin or BSD (B).

Western blot for PSD-95 from hippocampal neurons challenged with 5 M of A oligomers with or without dihydro-β-erythroidine (DHβE) an 42 nAChR inhibitor

(1M) (C). Each experimental group considers four independent experiments. Data is presented as means ± S.E.M. Statistical differences were calculated by Student´s t-test, followed by Dunnett’s post hoc test. Asterisks indicate statistically significant differences

(*p < 0.05). 2

Supplementary figure S2: Nicotine improves memory in wild-type mice. Comparison of the number of trials necessary to reach the criterion among wild-type and APP/PS1 transgenic animals treated with vehicle, or with nicotine on day 4. Representative track of

6-month-old wild-type control and nicotine-treated mice (A). Behavioral performance of young mice (6-month-old) was evaluated through a memory flexibility test, control (gray bar); nicotine treated (light gray bar) compared with transgenic control (white bar) or nicotine treated mice (black bar) (B). Each experimental group considers three animals.

Data is presented as means ± S.E.M. Statistical differences were calculated by ANOVA, followed by Bonferroni’s post hoc test. Asterisks indicate statistically significant differences (*p < 0.05).

Supplementary figure S3: Nicotine blocks Aβ aggregation in vitro. A: Kinetics of Aβ fibril formation (a) and disaggregation (b) followed in vitro by ThT fluorescence under constant stirring in absence (white circles), presence of 10 µM nicotine (black circles) or with 100 nM laminin for Aβ disaggregation assays as positive control (gray circle). B: The electron micrographs show A fibrils and A fibrils with nicotine (100 M) after 24 h of incubation (b). Bar represents 300 nm. Each experimental group considers three independent experiments. Data is presented as means ± S.E.M. Statistical differences were calculated by Student´s t-test, followed by Dunnett’s post hoc test. Asterisks indicate statistically significant differences (*p < 0.05). 3

Supplementary figure S4. α7-nAChR, PSD-95 and Synapsin-1 decrease in hippocampus of APP/PS1 in comparison with wild-type mice. Representative images of brain slices from wild-type mice and 12-month-old APP/PS1 mice stained for α7-nAChR,

PSD-95, and Synapsin-1 (A, B and C respectively). Quantification of CTCF in CA3 of 13 month-old APP/PS1 mice and wild-type mice are shown. Each experimental group considers three animals. Data is presented as means ± S.E.M. Statistical differences were calculated by ANOVA, followed by Bonferroni’s post hoc test. Asterisks indicate statistically significant differences (*p < 0.05).

SUPPLEMENTARY METHODS

Aggregation and Disaggregation assays. An aliquot of Apeptide stock solution (15

μg/µl) was added to aqueous buffer PBS pH 7.4. For the aggregation assay, 40 µM solutions was prepared from the Aβ stock solution added to a buffer containing 10 µM nicotine. For disaggregation assay, A fibrils were incubated with 10 M nicotine. The samples of aggregation and disaggregation were stirred continuously at room temperature at 1300 r.p.m. and sterile aliquots were taken at different time points for analysis. Aliquots were taken to measure A fibril formation by ThT fluorescence and A oligomers by

Western blot, as described previously (Toledo and Inestrosa, 2010).

Tht fluorimetric assay. Amyloid aggregates obtained were characterized by Th-T fluorimetric assay. Th-T binds specifically to amyloid fibrils, such binding produces a shift 4 in its emission spectrum, and an increase in the fluorescent signal, which is proportional to the amount of amyloid fibril formed. Following incubation of Aβ alone or co-incubated with nicotine, sodium phosphate buffer; 50 mM pH 6.0 and Th-T in (0.1 mM) were added up to a final volume of 2 ml. Fluorescence was monitored at excitation 450 nm and emission 485 nm using a JASCO FP-6200 Spectrofluorometer, as previously described

(LeVine, 1993).