EmoryAMPAD Proteomic Network Symposium BLSA Insoluble Proteome Network

Emory-AMP-AD Proteomic Network Symposium BLSA Insoluble Proteome Network: Mapping changes in the AD detergentinsoluble proteome Chad Hales M. D. , Ph. D. Assistant Professor of Neurology Emory University School of Medicine July 13, 2016

Approach • Brain homogenate – Total – Membrane – Soluble – Insoluble • Protocol – Gel, single shot, TMT

Why the detergent insoluble brain fraction? • Enriched proteins – Co- or post-translational folding – Degradation inefficiencies – Overall higher cellular expression • Reduced proteins – More effectively degraded – Preferential protein quality control mediated reductions in mis-folded states – Lower levels of cellular expression

AD insoluble proteome Homogenize Sarocosyl/Na. Cl/sonicate 200, 000 G spin Soluble Insoluble 8 M Urea Mass spectrometry Bai B, Hales C, Chen P, Gozal, Y et al. U 1 small nuclear ribonucleoprotein complex and RNA splicing defects in Alzheimer’s disease. (2013) PNAS.

A B A B A B AD /P ora D AD dic) (sp ora dic ) U 1 -70 k (sp D CB LD FT S AL PD CT -U RNA processing A B A APP Tau, isoform 4 Tau, isoform 5 Apo. E Clusterin Collagen α 1 -(XXV) U 1 -70 k U 1 A Sm B Sm D 1 Sm D 2 Sm D 3 Luc 7 -like DDX 46 / Prp 5 U 1 -70 k Tau C *

Evidence for RNA processing dysfunction in postmortem human brain 5’ RNA-seq Pre-m. RNA Exon 1 Exon 2 X U 6 U 4 U 1 U 5 5’ U 2 Exon 1 Nanostring Exon 1 5’ Exon 2 3’ 3’ m. RNA Bai B, Hales C, Chen P, Gozal, Y et al. U 1 small nuclear ribonucleoprotein complex and RNA splicing defects in Alzheimer’s disease. (2013) PNAS.

Other insights from the detergent insoluble fraction • AD continuum (pathology free controls, MCI, Asym. AD, AD) to understand how the RNA processing factors are changing • Familial AD secondary presenilin 1 mutations with comparison to late onset AD and transgenic rodents.

Approach Homogenize Sarocosyl/Na. Cl/sonicate 200, 000 G spin Soluble Insoluble

3 74 PS 1 EOAD 100 Asym. AD log 2 expression (PS 1 -Control) 723 R 2 = 0. 4477 2 1 -4 -2 0 0 2 4 6 -1 -2 -3 log 2 expression (Asym. AD-Control) 3 90 PS 1 EOAD 679 PS 1 EOAD 149 MCI 218 201 LOAD R 2 = 0. 5677 2 1. 5 1 0. 5 -4 0 -2 -0. 5 0 2 4 6 -1 -1. 5 log 2 expression (MCI-Control) log 2 expression (PS 1 -Control) 658 log 2 expression (PS 1 Control) 2. 5 -4 5 4 R 2 = 0. 7674 3 2 1 -2 0 -1 0 2 4 -2 -3 log 2 expression (LOAD-Control) 6

PS 1 -EOAD and APP/PS 1 mouse 708 PS 1 EOAD 189 752 APP/PS 1 -Ms log 2 expression (PS 1 -Control) 5 4 3 R 2 = 0. 0433 2 1 -2 0 -1 0 2 4 6 -2 -3 log 2 expression (APP/PS 1 Tg-WT) PS 1 -EOAD and APP/PS 1 Rat 843 PS 1 EOAD 54 520 APP/PS 1 -Rt log 2 expression (PS 1 -Control) 5 4 R 2 = 0. 1894 3 2 1 -4 -2 0 -1 0 2 4 -2 -3 -4 log 2 expression (APP/PS 1 Tg-WT) 6

Red p=6 e-04 Control LOAD Grey 60 p=6. 7 e-05 EOAD Magenta p=0. 0055 Control LOAD EOAD Violet p=0. 18 Control LOAD EOAD Black p=0. 00012 Control LOAD EOAD Green p=0. 015 Control LOAD EOAD Tan p=0. 0026 Control LOAD EOAD Darkgreen p=0. 39 Control LOAD EOAD Lighsteelblue 1 p=0. 56 Control LOAD EOAD

Complement proteins EOAD-LOAD enriched * * * RNA processing mitochondria

A. WT APP/ PS 1 1 2 3 littermates APP Tau Apo. E Clusterin U 1 -70 k U 1 A Sm B Sm D 1 C. H. D. I. Expression Level (Log 2) + 3. 0 mean - 3. 0 B. C. J. K. D. E. L. M. F. G. N. O.

Summary • Track disease progression in the detergent insoluble brain fraction • sn. RNP insolubility is happening relatively early in the disease cascade • Unique properties of LOAD and PS 1 -EOAD • Challenges with using transgenic animals to recapitulate all aspects of AD