A B C * 2 2.5 Aβ 1.8 n.s Aβ sAPPβ * Aβ 1.6 sAPPβ 2 n.s * 1.2 1.4 * 1 1.2 1.5 n.s n.s n.s 0.8 1 n.s 0.8 1 n.s 0.6 0.6 0.4 0.4 0.5
Normalized ECL Counts Normalized ECL 0.2
0.2 *** Counts Normalized ECL
Normalized ECL Counts Normalized ECL *** 0 0 0 *** MEDGC APP BACE1 Pen2 STX 7 VAMP-7 VAMP-5 PCDNA TFEB TFEB dNLS CONTROL 50uM 100uM Overexpression Chloroquine siRNA
1.2 D 1.2 E F AAV-TFEB injection into the brain cortical regions of WT mice
1 1 * 0.8 0.8 *
0.6 0.6
0.4 0.4 Normalized ECL counts Normalized ECL Normalized ECL counts Normalized ECL 0.2 0.2
0 0 PCDNA TFEB S211A/ PCDNA TFE3 S142A G H I
1.2 Aβ x-40 1.2 Aβ x-42 1.2 n.s 1 1
1 0.8 ** 0.8 *** 0.8 0.6 0.6 0.6 Values ed E CL
0.4 0.4 0.4 Normalized ECL counts Normalized ECL 0.2 0.2 0.2
Normali z *** 0 0 0 No CathepsinD+ CathepsinD+ Aß Heat inactivated Aß CathepsinD+ Aß GFP TFEB GFP TFEB
Mondal et al, SFig.1 1.4
1.2 Aß
1 sAPPß
0.8
0.6
0.4
0.2 Normalized ECL counts Normalized ECL 0 MEDGC EIF2AK1 APP BACE1 siRNA
Mondal et al., SFig.2 HeLa-swAPP cells A B 1.2 1.2 Aβ 1 1 sAPPβ Aβ 0.8 0.8 *
0.6 0.6 **
0.4 0.4
0.2 0.2 Normalized ECL counts Normalized ECL Normalized ECL counts Normalized ECL *** 0 0 MEDGC AKT1 AKT2 APP MEDGC AKT1 AKT2 APP siRNA siRNA
Mondal et al, SFig.3 B sAPPβ A Actin Synaptic Infection/ Endocytosis Lipid Metabolism Cytoskeleton Function
NRK NRK ROR1 HGS HGS STK35 WNK3 PLXNA3 CSK CSK MPP3 ROR1 RIPK3 CDK5 CDK5 STK11 CSK CSK PIP5K1B PIP5K1B CDK5 RYK EIF2AK1 CDK5 IRAK1
hsa04722:Neurotrophin signaling pathway hsa04910:Insulin/Nutrient pathway hsa04010:MAPK signaling pathway NRK STK32B CDC2L1 hsa04340:Hedgehog signaling pathway CNKSR1 VRK2 AKDH18A1 CMPK1 hsa04062:Chemokine signaling pathway NTRK2 CNKSR1 WNK3 BRSK1 HIPK2 hsa04620:Toll-like receptor signaling pathway CSK ROR1 CSK STK11 MPP3 EIF2AK1 hsa04070:Phosphatidylinositol signaling system RIPK3 CSK AURKA STRADB EIF2AK4 hsa04150:mTOR signaling pathway IRAK1 CDK5 hsa05200:Pathways in cancer Energy RNA processing/ hsa04360:Axon guidance NF B signaling Cancer Mental Health metabolism Translation hsa04920:Adipocytokine signaling pathway β hsa04370:VEGF signaling pathway APP hsa00230:Purine metabolism hsa00561:Glycerolipid metabolism hsa04916:Melanogenesis hsa04310:Wnt signaling pathway hsa04720:Long-term potentiation hsa00564:Glycerophospholipid metabolism Aβ hsa05020:Prion diseases hsa04810:Regulation of actin cytoskeleton Neurotrophin MAPK Hedgehog Synaptic PhosphatidylInositol hsa04110:Cell cycle Signaling Signaling Signaling Function Signaling hsa00983:Drug metabolism MAPKSP1 NTRK2 hsa00240:Pyrimidine metabolism IRAK2 NTRK2 TAOK2 TAOK2 PLXNA3 PIK3CG hsa00562:Inositol phosphate metabolism PIK3CG MAPK9 CSNK1A1 STK36 MAPK2 MAPK9 PTK2 DGKE hsa04020:Calcium signaling pathway IRAK1 MAPK7 CSNK1G1 CSNK1A1L MAP4K2 PRKACA PAK3 PIP5K1B hsa04140:Regulation of autophagy MAP2K2 CSK CSNK1D PRKACA STK3 MAPK7 PLXNA2 DGKH hsa00330:Arginine and proline metabolism PRKCD CALM1 GSK3B PRKACB MAP4K3 PRKACB GSK3B DGKI AKT1 AKT2 hsa00010:Glycolysis / Gluconeogenesis AKT1 MAP2K6 CDK5 PI4KB GSK3B MAP2K5 hsa04115:p53 signaling pathway MAP3K6 AKT2 CALM1 hsa04622:RIG-I-like receptor signaling pathway hsa04630:Jak-STAT signaling pathway hsa04740:Olfactory transduction NME2 PIK3CG AKT2 PIK3CG CSNK1A1 GK PIK3CG PRKACA PKM2 AKT1 MAPK9 AKT1 CSNK1A1L DGKE AKT1 PRKAA1 AK5 MAP2K2PRKACA AKT2 GSK3B ETNK2 AKT2 CSK DGUOK PHKG1 PRKAA1 STK11 MAPK9 DGKH GSK3A PRKCD AK7 GSK3B PRKACB ULK2 PRKACA DGKI GSK3B HCK PAPSS2 PRKC1 CALM1 PRKAA1 PRKACB PI4KB PRPS1 Insulin /Nutrient mTOR Chemokine Wnt Purine/ Signaling Signaling Signaling Signaling Glycerolipid/ Glycerophosholipid metabolism C
Mondal et al., SFig.4 AKT2 siRNA
1.4 oligo1 oligo2 oligo3 APP MedGC 1.2 Aβ 60 AKT2 1 GAPDH 0.8 38 kDa * 0.6 **
0.4 ** 0.2 *** *** *** Normalized ECL counts Normalized ECL *** 0 MED GC APP BACE1 PEN2 AKT2 AKT2 AKT2 AKT2 Oligo1 Oligo2 Oligo3 Pool
siRNA
Mondal et al., SFig.5 1.4 Aβ
1.2
1
0.8
0.6
0.4
0.2 Normalized ECL Counts Normalized ECL 0 MEDGC APP AKT1 AKT2 siRNA
Mondal et al, SFig.6 Mondal et al., SFig. 7 A B siRNA C Inhibitors MedGC APP BACE1 PEN2 AKT2 AKT1 DMSO C3 DAPT AKTinh1 Lo AKTinh 1 Hi AKT2inhII Lo AKTinhII Hi DMSO DAPT AKT inh
98 APP 98 APP
β-CTF β-CTF (C99) 14 14 -CTF -CTF (C83)
38 GAPDH 38 GAPDH kDa kDa
Mondal et al, SFig.8 C 1.4 1.4 B 1.4 Aβ n.s A Aβ Aβ sAPPβ n.s 1.2 n.s n.s 1.2 1.2 1 1 1 0.8 ** * 0.6 0.8 0.8 ** 0.4 ***
0.6 0.6 ** Normalized to DMSO 0.2 ****** ** 0.4 0 0.4 ****** *** *** DMSO AKT pathway AKT pathway AKT pathway inhibitor 10 M inhibitor 5 M inhibitor 1 M
Normalized ECL counts Normalized ECL 0.2 Normalized ECL counts Normalized ECL 0.2 *** *** 0 0 C3 DAPT DAPT PI-103 DMSO Torin-1 Torin-1 DMSO AZD2014 Everolimus KU-0063794 AKT-Inh. VIII AKT-Inh. Perifosine-Hi Perifosine-Lo Miltefosine-Hi Miltefosine-Lo AKT pathway Inhibitors PH-domain targeting AKT Inhibitors SH-SY5Y Cells expressing endogenous APP Aβ D 1.2 E 1.2 Aβ
1 1
0.8 0.8 *** 0.6 0.6 *** 0.4 0.4
0.2 0.2 *** Normalized ECL counts Normalized ECL Normalized ECL counts Normalized ECL *** 0 0 DMSO AKT Inhibitor DAPT DMSO AKT Inhibitor DAPT
Mondal et al, SFig.9 A
B
NESTIN/ DACH1 PHASE CONTRAST
SOX2 beta III-tubulin/ GFAP
Mondal et al, SFig.10 4000 Aβ 40 3500 * WT AKT2 -/- 3000 2500 60 2000 AKT2 1500 1000 Normalized ECL counts Normalized ECL 500 38 GAPDH 0 kDa WT AKT2 -/-
Mondal et al, SFig.11 Aβ A sAPPβ
Supernatants from Cells that Supernatants from Cells that overexpress APP do not overexpress APP