PDK1-dependent quenching of TACE shedding activity in prion and Alzheimer s diseases Mathéa Pietri, Caroline Dakowski, Samia Hannaoui, Aurélie Alleaume-Butaux, Julia Hernandez-Rapp, Audrey Ragagnin, Sophie Mouillet-Richard, Stéphane Haik, Yannick Bailly, Jean-Michel Peyrin, Jean-Marie Launay, Odile Kellermann, and Benoit Schneider Supplementary Fig. 1 Supplementary Figure 1: Accumulation of PrP Sc in primary cerebellar granule neurons and in the brain of mice infected by the 22L strain. (a) Western blotting to show kinetics of PrP Sc accumulation in primary cerebellar granule neurons (CGNs) exposed to brain homogenate of terminally ill 22L-infected mice (22L-CGN) up to 28 days after infection (d). As a control, CGNs were exposed to brain homogenate of healthy mice (CGN). (b) Western blotting to reveal PrP Sc in brain extracts of 22L-infected mice (22L) as compared to Sham-inoculated mice (Sham).
To discriminate between PrP C and PrP Sc, CGNs or brain extracts were treated with proteinase K (PK) and immunoblotted with SAF83 PrP antibody for PrP res detection. Shown are representative Western blots of three independent experiments.
Supplementary Fig. 2 1C11 50 ng ml 1 stnf-! " +! + +! BX912! +! +! Fk-1C11! 22L-1C11! + +! +! 15 kda! Actin! Active caspase 3! 37 kda! 10 ng ml 1 stnf-! " BX912! 15 kda! 37 kda! 1C11 5-HT Fk-1C11 5-HT! 22L-1C11 5-HT! +! + +! + +! +! +! +! Active caspase 3! Actin! 50 ng ml 1 TNF-! induced! caspase-3 activation! 6! 4! 2! 0! 1C11!!"!"!!"!!" +! +! Ctrl! Fk! 22L! 10 ng ml 1 TNF-! induced! caspase-3 activation! 8 6 4 2 0 BX912! (1 µm)! 1C11 5-HT!"!!!"!"!!" +! +! Ctrl! Fk! 22L! Supplementary Figure 2: Prion infection exacerbates stnf-!-induced caspase-3 activation, which is counteracted upon PDK1 inhibition. Western blotting to show activation of TNFR1-coupled caspase-3 in Fk- or 22L-infected 1C11 and 1C11 5-HT cells exposed to stnf-! for 120 min vs. non-infected cells (Ctrl). PDK1 inhibition with BX912 (1 µm, 3 h) in infected cells restores pre-infectious cell sensitivity to stnf-!. Quantitative data are shown as the mean ± SEM from three experiments performed in triplicate. # P < 0.005 vs. non-infected cells exposed to stnf-! (Ctrl); ## P < 0.01 vs. Fk- or 22L-infected cells treated with stnf-!.
Supplementary Fig. 3 Supplementary Figure 3: Prion infection causes brain TNFR1 accumulation. Immunostainings of TNFR1 (a) or PrP Sc (b) in the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) of 6PB1- and 22L-infected mice at 90 days after infection with 6PB1 strain and 130 days after infection with 22L strain vs. Sham-inoculated mice. Note that TNFR1 and PrP Sc stainings of CBCX molecular layer of 22L-infected mice exhibit a banded patterning (arrows), suggesting that the increased amount of TNFR1 topographically correlates with PrP Sc deposits. All experiments were performed with n = 3 mice for each condition. Images of one representative experiment are shown. Scale bar = 100!m.
Supplementary Fig. 4 a! b! c! 1C11! B! saponin 0.05%!! + Fk-1C11! FAK! Cav-1! TACE! saponin! 120! Intensity of TACE! signal (% of 1C11)! 100! 80! 60! 40! 20! 0!! +! + 1C11! Fk-1C11! Fraction! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! 12! 125 kda! 21 kda! 100 kda! 100 kda! 100 kda! 100 kda! Cell surface biotinylation! IP: TACE! WB: streptavidin! 100 kda! #! * 1C11 5-HT! 1C11 5-HT! 1C11 5-HT! CGN! d! Fk-1C11 5-HT! Fk-1C11 5-HT! +BX912! Fk-1C11 5-HT! +anti-cav-1! 1C11 5-HT! kda! 100! 100! 100! 100! 100! 100! 100! 100! 100! 22L-CGN! 6! 5! Intensity of TACE! signal (% of ctrl)! ph! 120! 100! 80! 60! 40! 20! 0! 1C11! Fk-1C11! Fk-1C11+BX912! 1C11 5-HT! Ctrl! Fk-1C11 5-HT! Fk-1C11 5-HT +BX912! CGN! 22L-CGN! CGN! #! 22L! 22L-CGN+BX912! Fk-1C11 5-HT! 100 kda! Fk-1C11 5-HT! +BX912, 37 C! 100 kda! Fk-1C11 5-HT! TACE! pi 5.8! p~tace! 4.9 < pi < 5.1! 100 kda! +BX912, 4 C! e! Non-infected! 1C11 5-HT! Fk-1C11 5-HT! + PP2! + Wortmannin! + BX912! + PD98059! Supplementary Figure 4: PDK1 overactivity promotes TACE phosphorylation and internalization in prion-infected cells.
(a) Immunofluorescent labeling to assess TACE presence at the surface of non-infected 1C11 cells and primary CGNs, and their infected counterparts (Fk-1C11; 22L-CGN). Cell permeabilization with saponin reveals that TACE is internalized in Fk-infected 1C11 cells. Note that the intensity of TACE signal measured in permeabilized infected cells is comparable to that measured at the surface of non-infected cells. (b) Immunoblot analysis of sucrose gradient fractions of 1C11 5-HT cell membranes to reveal that (i) in non-infected cells TACE co-segregates with FAK, thus indicating that TACE is present at the plasma membrane; (ii) in Fk-infected cells, TACE shifts into caveolin-1 (Cav- 1)-enriched fractions; (iii) treatment with a PDK1 inhibitor (BX912, 1!M for 1 h) or immunosequestration of Cav-1 (anti-cav-1) in Fk-infected cells relocate almost all TACE molecules at the plasma membrane. (c) Sucrose gradient fractionation after cell surface protein biotinylation to reveal that (i) in non-infected 1C11 5-HT cells, TACE is present at the plasma membrane; (ii) in Fk-1C11 5-HT cells, TACE is not present at the plasma membrane; (iii) treatment with a PDK1 inhibitor (BX912, 1!M for 1 h) at 37 C relocates TACE molecules to the plasma membrane. Temperature block at 4 C counteracts the BX912-induced relocation of TACE to the plasma membrane. (d) 2D-gel electrophoresis to assess TACE phosphorylation in Fk-infected 1C11, 1C11 5-HT cells and 22L-infected CGNs treated or not with BX912 (1!M for 1 h) vs. non-infected cells. In non-infected cells, TACE displays an apparent molecular mass of ~100 kda and a pi close to 5.8, corresponding to weakly phosphorylated TACE (www.phosphosite.org). Upon infection, part of TACE spots shifted to a more acidic ph (pi of 5.1 to 4.9) without any measurable change of the apparent molecular mass (arrows). Exposure of infected cells to BX912 cancels prion-induced TACE phosphorylation.
(e) Immunofluorescent labeling to show TACE presence at the surface of Fk-1C11 5-HT cells treated or not with inhibitors of Src kinases (PP2, 1!M), PI3K (Wortmannin, 1!M), PDK1 (BX912, 1!M) or the MEK-ERK1/2 module (PD98059, 5!M) for 1 h vs. non-infected cells. All experiments were performed three times in triplicate. * P < 0.005 vs. non-permeabilized infected cells; # P < 0.005 vs. non-infected cells (Ctrl); Scale bar = 50!m.
Supplementary Fig. 5 a! b! stnfr1 (pg ml 1 )! PrP Sc amount! (ng per mg protein)! 60! 50! 40! 30! 20! 10! 0! 12! 10! 8! 6! 4! 2! 0! 1C11 5-HT! #! #! * * + + +! Anti-Cav-1! Ctrl! Fk! 22L! 1C11 5-HT! #! #! + +! Anti-Cav-1! Fk! 22L! Supplementary Figure 5: Caveolin-1 blockade rescues TNFR1 shedding and reduces PrP Sc accumulation in prion-infected neurons. (a) ELISA stnfr1 quantifications in the culture medium of Fk- and 22L-infected 1C11 5-HT cells vs. non-infected 1C11 5-HT cells (Ctrl) to assess the impact of Cav-1 immunosequestration (anti-cav-1) on TNFR1 shedding. (b) ELISA PrP res quantifications to assess the impact of Cav-1 blockade (anti-cav-1) on PrP Sc level in Fk- and 22L-infected 1C11 5-HT cells.
All experiments were performed three times in triplicate. * P < 0.01 vs. non-infected cells (Ctrl); # P < 0.01 vs. untreated Fk- or 22L-infected cells.
Supplementary Fig. 6 a! Cell surface TNFR1! level (fold increase)! 2.5! 1.5! 0.5! TgA! neg! TgA! pos! 0! 2! 1! #! TgA! neg! TgA! pos! b! Cell culture medium stnfr1 level (ng ml 1 )! 70 60 50 40 30 20 10 #! * c! 0 BX912! +! +! TgA! neg! TgA! pos! Fraction! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! 12! 100 kda! TgA! neg! TACE! 100 kda! 100 kda! 100 kda! TgA! pos! TgA! pos! +BX912! TgA! pos! +PD98059! Cav-1 positive fractions! FAK positive fractions! Supplementary Figure 6: TNFR1 under-shedding in hippocampal AD neurons originates from PDK1-dependent dysregulation of TACE sheddase activity. (a) Immunofluorescent labeling to show TNFR1 presence at the surface of TgA! pos neurons vs. TgA! neg neurons. Scale bar = 50!m. For each group n = 4. # P < 0.05 vs. TgA! neg neurons. (b) ELISA stnfr1 quantifications in the culture medium of TgA! pos vs. TgA! neg neurons treated or not with BX912 (1!M for 1 h). For each group, individual values and median (bar) are shown. # P < 0.05 vs. TgA! neg neurons; * P < 0.05 vs. untreated TgA! pos neurons.
(c) Immunoblot analysis of sucrose gradient fractions of Tg2576-derived hippocampal neuron membranes to reveal that (i) in TgA! neg neurons TACE co-segregates with FAK, indicating that TACE is present at the plasma membrane prior to A! plaque deposition; (ii) in TgA! pos neurons, TACE shifts into caveolin-1 (Cav-1)-enriched fractions; (iii) treatment of TgA! pos neurons with BX912 (1!M for 1 h) relocates most of TACE molecules to the plasma membrane; (iv) inhibition of the MEK-ERK1/2 module with PD98059 (5!M for 1 h) in TgA! pos neurons fails to target TACE back to the plasma membrane. For each group n = 4.
Supplementary Fig. 7 Supplementary Figure 7: PDK1 inhibition durably reduces A! plaque burden in the hippocampus and cortex of Tg2576 mice. Intraperitoneal infusion of BX912 (5 mg kg!1 per day; 0.25 "l per h) to 200 days-old Tg2576 mice reduces the number of animals that accumulate A! (BX912-treated TgA! pos mice) to 10/100 vs. 23/100 at 300 days and to 12/100 vs. 34/100 at 330 days as compared to untreated mice. As observed at 275 days (Fig. 6a), each of these BX912-treated TgA! pos animals displays a ~2-fold reduced number (a) and surface (b) of amyloid plaques in both the hippocampus (Hp) and cortex (Cx) as compared to untreated TgA! pos animals at 300 and 330 days. # P < 0.001 vs. untreated TgA! pos animals.
Supplementary Fig. 8 Supplementary Figure 8: PDK1 silencing in Tg2576 mice rescues cognition and memory. Contextual fear conditioning test with 275 days-old TgA! pos mice to assess the impact of sirna-mediated PDK1 silencing (sipdk1) starting at day 265 on cognition and memory vs. non-treated TgA! pos and TgA! neg mice. A scramble sirna (siscr) is used as control. For each group, individual values and median (bar) are shown. * P < 0.01 vs. TgA! neg mice; ## P < 0.05 vs. siscr-treated TgA! pos mice.
Supplementary Fig. 9 Supplementary Figure 9: PDK1 inhibition in Tg2576 mice improves hippocampal functions.
Morris water maze test to assess the impact of PDK1 inhibition in TgA! pos mice on hippocampal functions. BX912 treatment improves the latency to reach the platform during the trial period (a), the number of crossing (the mice crossed the position where the platform was placed during trial sessions) (b), the percent time spent in NW quadrant (in which the platform was during the trial period) as compared to other quadrants (c). Note that the swimming speed keeps constant around 20 cm per s for all tested animals (n = 8 for each group). Values are means ± SEM. * P < 0.01 vs. TgA! neg mice; ## P < 0.01 vs. untreated TgA! pos mice.
Supplementary Fig. 10 Supplementary Figure 10: PDK1 inhibition in 3Tg mice alleviates Alzheimer s disease.
(a) Impact of intraperitoneal injection of BX912 (5 mg kg!1 per day; 0.25 "l per h) in 300 days-old 3TgA! pos mice for 75 days on APP processing by measuring CSF concentrations of sapp", sapp!, A!40 and A!42 vs. untreated 3TgA! pos and 3TgA! neg mice. For each group, individual values and median (bar) are shown. * P < 0.05 vs. 3TgA! neg mice; ## P < 0.05 vs. untreated 3TgA! pos mice. (b-d) Contextual fear conditioning task (b), Morris water maze test (c) and nest construction (d) to assess the impact of PDK1 inhibition on memory and cognition deficits in 3TgA! pos mice vs. untreated 3TgA! pos and 3TgA! neg mice. For each group, individual values and median (bar) are shown. * P < 0.01 vs. 3TgA! neg mice; ## P < 0.01 vs. untreated 3TgA! pos mice.
Supplementary Fig. 11 Supplementary Figure 11: PDK1 inhibition in 5Tg mice alleviates Alzheimer s disease.
(a) Impact of intraperitoneal injection of BX912 (5 mg kg!1 per day; 0.25 "l per h) in 140 days-old 5TgA! pos mice for 75 days on APP processing by measuring CSF concentrations of sapp", sapp!, A!40 and A!42 vs. untreated 5TgA! pos and 5TgA! neg mice. For each group, individual values and median (bar) are shown. * P < 0.05 vs. 5TgA! neg mice; ## P < 0.05 vs. untreated 5TgA! pos mice. (b-d) Contextual fear conditioning task (b), Morris water maze test (c) and nest construction (d) to assess the impact of PDK1 inhibition on memory and cognition deficits in 5TgA! pos mice vs. untreated 5TgA! pos and 5TgA! neg mice. For each group, individual values and median (bar) are shown. * P < 0.01 vs. 5TgA! neg mice; ## P < 0.01 vs. untreated 5TgA! pos mice.
Supplementary Fig. 12 Supplementary Figure 12: Schematic representation of TACE dysregulation caused by PDK1 overactivity in prion-infected or AD neurons and the beneficial effects of PDK1 inhibition against prion or Alzheimer s diseases. Prion infection or the accumulation of A! peptides causes the overstimulation of the Src kinases/pi3k/pdk1 signaling pathway. Overactivated PDK1 promotes the phosphorylation and internalization of TACE metalloproteinase, which diverts the processing of PrP C, APP and TNFR1 away from the TACE "-secretase action. Defect of TACE sheddase activity at the plasma membrane of prion-infected or AD neurons contributes to the conversion of PrP C into PrP Sc or the production of pathogenic A! peptides, and the overexposure of TNFR1, which renders prion-infected or AD neurons highly sensitive to TNF-" toxicity. PDK1 inhibition with BX912 or sirna permits TACE to dephosphorylate and to relocate to the plasma membrane, where it recovers its "-cleavage activity towards PrP C, APP and TNFR1.
Rescue of TACE activity at the plasma membrane upon PDK1 inhibition (i) desensitizes pathological neurons from TNF-" toxicity upon TNFR1 shedding and the release of stnfr1, (ii) limits the conversion of PrP C into pathogenic PrP Sc in prion-infected neurons upon "- cleavage of PrP C and (iii) counteracts the production of neurotoxic A! peptides in AD neurons upon "-cleavage of APP. Therefore, PDK1 emerges as a novel therapeutic target for prion and Alzheimer s diseases.
Supplementary Table 1 stnf-! LD 50 (ng ml!1 ) Non-infected Fk-infected 22L-infected 1C11 70 ± 10 13.0 ± 1.5 9.0 ± 1.0 1C11 5-HT 8.1 ± 1.2 2.0 ± 0.7 4.0 ± 0.2 CGN 100 ± 20 2.6 ± 0.6 3.1 ± 1.4 Supplementary Table 1: Impact of prion infection on cell sensitivity to stnf-! in 1C11 precursor cells, their 1C11 5-HT neural derivative and primary CGNs. stnf-! LD 50 values correspond to the concentration of stnf-! inducing a 50% cell death in 1C11 and 1C11 5-HT cells or inducing dendritic fragmentation for 50% of neuronal cells in CGNs. Data are the mean ± SEM of three independent experiments performed in triplicate.
Supplementary Table 2 Sample ID Age Gender PMI Braak AD1 78 F 5 6 AD2 85 F 9 5 AD3 82 M 6 6 AD4 69 F 9 5 AD5 87 M 5 5 AD6 92 M 8 6 C1 75 F 6 1 C2 87 F 5 1 C3 80 M 9 1 C4 70 F 6 2 C5 84 M 8 2 C6 87 M 8 1 Supplementary Table 2: Selected patients. Patients were divided in two groups according to neuropathological and clinical criteria: AD patients (AD) and non-ad patients (C). Sample identification (ID); Age at death; female (F); male (M); post-mortem interval in hours (PMI); Braak stage.