Supporting Material

IRE1 signaling exacerbates Alzheimer's disease pathogenesis

Claudia Duran-Aniotz1,2,3*, Victor Hugo Cornejo1,2,3*, Sandra Espinoza1,2,3, Álvaro O. Ardiles4, Danilo B. Medinas1,2,3, Claudia Salazar4, Andrew Foley1,3, Ivana Gajardo4,Peter Thielen5, Takao Iwawaki6,Wiep Scheper8,9,Claudio Soto7, Adrian G. Palacios4, Jeroen M. Hoozemans10, and Claudio Hetz1,2,3,5,11#.

#Address correspondence to:

Claudio Hetz: Institute of Biomedical Sciences (Sector B, second floor) University of Chile. Independencia 1027, Santiago, Chile, P.O.BOX 70086, Tel +56-229786506 email: or .

Supplementary figures

Supplementary Fig. S1 XBP1s expression levels increase in human AD samples.a XBP1s mRNA levels were quantified in controls and late AD stages (V-VI) using Real-time qPCR. Data were represented as XBP1s/XBP1 total levels (n = 17 per group). bDouble immunolabeling for Aβ clone IC16 (red) with p-IRE1 (brown) in the CA1 region of AD cases (Braak VI, higher magnification right panel). Scale bar: 200 µm (upper panel) and 50 µm (lower panel).Values in aare expressed as means ± SEM. Data were analyzed by Student's t-test. *: p < 0.05.

Supplementary Fig.S2Generation of 5xFAD mice with conditional deletion of IRE1 in the nervous system.a IRE1cKO mice were crossed with 5xFAD animals to generate experimental animals and conventional PCR using genomic DNA was performed to determine the different genotypes. ThS distribution was determined in serial sections from the entire cortex and hippocampus regions of animals presented in b and c. ThS deposits were quantified in cortical d and hippocampal e areas of 5xFAD and IRE1cKO/5xFAD at 8 month of age. Values are expressed as means ± SEM. 5xFAD (n = 6-15) and IRE1cKO/5xFAD (n = 6-9) animals.Data was analyzed by Student's t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Supplementary Fig.S3Control measurements in the Morris water maze behavioral test. Experimental animals presented in Fig. 4were analyzed to measure possible motor and visual problems using water maze behavioral test. Average speed and number of entries were evaluated at fifth day of the behavioral paradigm in all experimental animals. Values are expressed as means ± SEM.

Supplementary Fig. S4Aggregation of human mutant APP in 5xFAD mice. a Detection of aggregated species of hAPP in brain cortex of 5xFAD mice by filter trap and centrifugal sedimentation. b Analysis of hAPP protein aggregates in brain cortex of 8-month old 5xFAD and IRE1cKO/5xFAD animals. hAPP levels were quantified using β-actin as loading control. Values are expressed as means ± SEM. Data was analyzed by Student's t-test using for statistical significance: *: p0.05.

Supplementary Fig.S5 XBP1 expression controls APP levels.aNeuro2a cells were stably transduced with lentiviral vectors expressing shRNA against Xbp1 or control luciferase mRNA (shXBP1 and shLUC, respectively), and expression of XBP1s was analyzed by western blot after treatment with Tm (1 μg/mL) for 16 h.b shLUC and shXBP1 cells were transiently co-transfected with APP-Flag expression vector and empty (pcDNA3) vector or XBP1s construct. After 48 h of expression, cells were lysed for Western blot analysis using anti-Flag antibody.cNeuro2a shLUC and shXBP1 cells were transiently transfected with APP-GFP expression vector. Cells were re-plated 24 h post-transfection and after 48 htreated with a lysosomal inhibition cocktail (200 nM Bafilomycin A1, 10 μg/mL Pepstatin and 10 μg/mL E64D, Lys Inh.) for 6 h. Then cells were lysed for Western blot analysis using anti-GFP and anti-p62 antibodies. Fold change of total APP-GFP levels relative to each control cell line is presented in the right panel. dSame as c, treating cells with the PI-3 kinase inhibitor 3-methyladenide(3-MA; 10 mM). ep62 and LC3-IIlevels were detected in brain cortex of 5xFAD and IRE1cKO/5xFAD animals at 6 months old to measure autophagy activation. As a positive control, spinal cord tissue of SOD1G85R transgenic mice was employed.

Supplementary Fig.S6IRE1 deficiency does not affect PERK, ATF6 signaling or BDNF levels in 5xFAD mice. aPhosphorylated eIF2levels were analyzed by Western blot in brain cortex of 5xFAD and IRE1cKO/5xFAD animals (n = 7 per group).bAs positive control for detection of phospho-eIF2, murine embryonic fibroblasts (MEF) were treated with increasing concentrations of the ER stressor tunicamycin (Tm) for 16 h and analyzed by Western blot in parallel. β-actin and GAPDH were employed as loading control.c ATF6 levels were analyzed by Western blot in brain cortex of 5xFAD and IRE1cKO/5xFAD animals (n = 3 per group). As positive control N2a cells treated with tunicamycin (Tm) for 16 h were also analyzed. ATF6f (active fragment) were detected in N2a cells. d Real time qPCR analysis of Bdnf mRNA levels in brain cortex and hippocampus (Hipp) of 5xFAD and IRE1cKO/5xFAD animals (n = 5-6 per group). Values are expressed as means ± SEM.Data was analyzed by Student's t-test for statistical significance.