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 ** Values 0.8 0.8 CL *** 0.6 E 0.6 0.6 ed z 0.4 0.4 0.4 Normalized ECL counts Normalized ECL 0.2 0.2 0.2 Normali *** 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 NFB 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 AKT inhibitor 1.2 1.4 DMSO AKT pathway Inhibitor 1.2 1 1 0.8 0.8 0.6 0.6 0.4 DMSO Normalized ECL counts Normalized ECL Normalized ECL counts Normalized ECL 0.4 Aβ sAPPβ 0.2 AKT pathway Inhibitor 0.2 0 0 0 Hr 2 Hr 4 Hr 6 Hr 0 Hr 2 Hr 4 Hr 6 Hr Time of incubation Time of incubation B Aβ sAPPβ C Supernatants from Cells that do not Cells that overexpress APP 1.4 Aβ overexpress APP 1.4 sAPPβ AKT inhibitor SEAP 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 Normalized ECL Counts Normalized ECL 0.2 0.2 Norm. Luminescence counts 0 0 DMSO AKTinh I Lo AKTinh I Hi AKT inh II Lo AKT inh II Hi MEDGC AKT1 AKT2 AKT inhibitor or DMSO treatment on cells that do not overexpress APP siRNA Mondal et al., SFig.12 A B Insulin Insulin 1.4 * 1.2 DMSO 0.5 1 1 98 APP (Low exposure) 0.8 0.6 * * * * 98 APP (High exposure) 0.4 0.2 Normalized ECL counts Normalized ECL 0 38 GAPDH DMSO AKT pathway DMSO+Insulin AKT pathway inhibitor Inhibitor+Insulin kDa C DMSO pathway inhibitor AKT pathway inhibitor AKT + Insulin DMSO + Insulin 98 APP 38 GAPDH kDa Mondal et al, SFig. 13 A B C Insulin + DAPI TFEB Merge Amino acids - + - + AKT inhibitor - - + + AKT pathway inhibitor - + DMSO TFEB S211-P Lamp2 TFEB GFP Tubulin AKT Inhibitor AKT mTOR *** Merge + DMSO AKT AKT- pathway Pathway inhibitor Inhibitor Mondal et al. SFig.14 6 5 4 Control 3 AKT 2 pathway inhibitor Fold changes (mRNA) relative to GAPDH 1 0 Lamp1 Lamp2 CatD vATPase Mondal et al, SFig.15 A B Insulin/IGF-1/Nutrient Signaling Protein synthesis Protein degradation (Autophagic/ Lysosomal) Protein accumulation In division competent cells, Failure to dilute proteins through this leads to cell division division in postmitotic neurons (Cancer) Protein aggregation (amyloid formation in Neurodegeneration) C Nutrient+Insulin Starvation 10-5 10-10 Pa nPa 10-11 10-12 Pa 10-10 nM nM nPa 10-13 10-14 10-15 10-15 0123 4 5 0123 4 5 time (days) time (days) 105 1.2 P P 1 m 104 m Pa Pa nPa nPa 0.8 103 0.6 102 0.4 fold change fold change 1 10 0.2 100 0 0123 4 5 0123 4 5 Mondal et al, SFig.16 A B C CHO wt NPC1 null NPC1 null_hNPC1 CHO wt NPC1 null NPC1 null_hNPC1 CHO wt NPC1 null NPC1 null_hNPC1 LysoTracker ThioS Filipin DMSO DMSO DMSO AKT-pathway inhibitor AKT-pathway inhibitor AKT-pathway inhibitor Mondal et al, SFig.17 A CHO wt NPC1 null NPC1 null_hNPC1 CHO wt NPC1 null NPC1 null_hNPC1 B Mondal et al, SFig.18 HeLa sw APP cells Supernatant Measure Aβ from the supernatant Seeding of Treatment 12h incubation Medium change BV2 microglia Mondal et al, SFig.19 A No metabolic disorder Hyperlipidemia T2D Control AD Synapsin-1, CD68, Iba1, DAPI B AD Hyperlipidimia Synapsin-1, CD68, Iba1, DAPI AD T2D Synapsin-1, CD68, Iba1, DAPI Mondal et al, SFig.20 High Insulin/ Nutrient Low Insulin/ Nutrient conditions conditions Amino Insulin APP BACE Insulin Amino acids γ-secretase acids Insulin Receptor Insulin Receptor PI3K PI3K AKT AKT Phospho- Aβ Phospho- TSC1/2 TSC1/2 TSC1/2 Early endosomes mTOR mTOR TSC1/2 Amino Amino TFEB-P/ acids acids TFE3 (Cytoplasm) TFEB/ TFE3 (nucleus) More functional Lysosomes Less functional Lysosomes More clearance of Less clearance of endosomally generated Aβ endosomally generated Aβ High Aβ Low Aβ High amyloid levels Low amyloid levels High Autophagolysosome Cholesterol ? ? Autophagosome Cathepsin D Golgi Mondal et al, SFig.21 Reactome(Pathway(Enrichment(Analysis( Pathway(name( FDR( Signaling'by'Interleukins' 3.75E612' Diseases'of'signal'transduction' 1.72E610' Signaling'by'SCF6KIT' 2.59E610' Insulin'receptor'signalling'cascade' 3.04E610' Signaling'by'VEGF' 3.04E610' Signaling'by'EGFR' 3.04E610' PIP3'activates'AKT'signaling' 3.04E610' IRS6mediated'signalling' 3.04E610' Negative'regulation'of'the'PI3K/AKT'network' 3.04E610' Signaling'by'Insulin'receptor' 3.05E610' IRS6related'events'triggered'by'IGF1R' 3.05E610' PI3K/AKT'activation' 3.05E610' GAB1'signalosome' 3.05E610' IGF1R'signaling'cascade' 3.05E610' Signaling'by'Type'1'Insulin6like'Growth'Factor'1'Receptor'(IGF1R)' 3.19E610' Downstream'signal'transduction' 3.90E610' VEGFA6VEGFR2'Pathway' 4.34E610' Signaling'by'PDGF' 4.34E610' DAP12'signaling' 4.34E610' Downstream'signaling'events'of'B'Cell'Receptor'(BCR)' 6.34E610' PI5P,'PP2A'and'IER3'Regulate'PI3K/AKT'Signaling' 6.34E610' DAP12'interactions' 9.63E610' MAPK1/MAPK3'signaling' 1.22E609' PI3K/AKT'Signaling'in'Cancer' 1.22E609' ' ' ! ! ! ! ! Mondal!et!al,!STable!1! Legends to Supplementary Figures Supplementary Figure 1: Replication and validation of Aβ clearance by lysosomes A.