Session MOF. There are 4 abstracts in this session.


The Human Brainome: Genome, Transcriptome and Proteome Interaction in Human Cortex

Amanda Myers

Our hypothesis is that changes in gene and protein expression are crucial to the development of late onset Alzheimer’s disease. Previously we examined how DNA alleles control downstream expression of RNA transcripts and how those relationships are changed in late onset Alzheimer’s disease. We have now examined how proteins are incorporated into networks in two separate series and evaluated our outputs in two different cell lines. Our pipeline included the following steps: 1. Predicting expression quantitative trait loci, 2. Determining differential expression, 3. Analyzing networks of transcript and peptide relationships and 4. Validating effects in two separate cell lines. We performed all our analysis in two separate brain series to validate effects. Our two series included 345 samples in the first set (177 controls, 168 cases; age range 65-105; 58% female; KRONOSII cohort) and 409 samples in the replicate set (153 controls, 141 cases, 115 mild cognitive impairment; age range 66-107; 63% female; RUSH cohort). Our top target is Heat Shock Protein Family A Member 2 (HSPA2), which was identified as a key driver in our two datasets. HSPA2 was validated in two cell lines, with overexpression driving further elevation of Abeta40 and Abeta42 levels in amyloid precursor protein mutant cells as well as significant elevation of microtubule associated protein Tau (MAPT) and phospho-Tau in a modified neuroglioma line. This work further demonstrates that studying changes in gene and protein expression is crucial to understanding late onset disease and further nominates HSPA2 as a specific key regulator of late onset Alzheimer’s disease processes.
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An Integrated Proteomics Approach to Resolve Brain-based Biomarkers in Alzheimer’s Disease

Nicholas Seyfried

There is a need for novel biomarkers of Alzheimer’s disease (AD) and other neurodegenerative disorders that are minimally invasive and that more broadly serve as accurate indicators of the underlying pathophysiological processes in brain. The Accelerating Medicine Partnership AMP-AD target discovery consortium is performing large scale multi-omics profiling and systems level integration of more than 2,000 postmortem human brains, establishing an unprecedented understanding of the pathophysiological processes driving cognitive decline, pathological burden, and other disease traits. The Emory AMP-AD team has focused on large scale proteomic analyses using unbiased label-free and isobaric tandem mass tag (TMT) based mass spectrometry methods to quantify thousands of proteins in brains from several different cohorts. Systems based network approaches reveal highly conserved modules of co-expressed proteins, many of which correlate strongly with clinical and pathological phenotypes, including those reflecting key mechanisms strongly correlated with impaired neuronal and synaptic function, neuroinflammation, and neurodegeneration. Preliminary studies were also performed to determine whether hub proteins representing these brain-based modules are found in cerebrospinal fluid (CSF). Following albumin depletion, we analyzed CSF samples from well-characterized AD and control patients and reliably quantified ~3,000 proteins by TMT based mass spectrometry across all samples. Of these, ~70% of the proteins were also identified in brain tissue, including members of phenotype-associated modules. Hence, large-scale proteomics with systems analyses provides a comprehensive dataset of brain-based protein changes linked to AD. This establishes a pipeline for targeting brain-based proteins in CSF as biomarkers for diagnosis, staging and therapeutic
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Proteogenomic analysis reveals FUS gene as dual-coding with both proteins united in molecular hallmarks of amyotrophic lateral sclerosis

Marie A Brunet; Jean-Francois Jacques; Amina Lekehal; Xavier Roucou
Université de Sherbrooke, Sherbrooke, Canada

The emergence of proteogenomics highlighted that current genome annotations do not capture the full coding potential of eukaryotic genomes. Novel coding sequences (alternative ORFs, altORFs) are camouflaged in “non-coding” RNAs, UTRs of mRNA or within annotated sequences in an alternative reading frame. We built OpenProt, a proteogenomic resource, which retrieves experimental evidence of altORFs from large-scale mass spectrometry (MS) and ribosome profiling data to better define proteomes.

Using OpenProt, we discovered a conserved altORF, named altFUS, nested in the FUS CDS; thus demonstrating the dual-coding nature of the Amyotrophic Lateral Sclerosis (ALS)-associated FUS gene. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons of healthy controls and ALS patients. AltFUS inhibits autophagy, a pathological hallmark presently and incorrectly attributed to the FUS protein. AltFUS is also pivotal in the loss of mitochondrial membrane potential and accumulation of FUS/TDP-43 cytoplasmic aggregates. Suppression of altFUS expression in a FUS-ALS Drosophila model protects against neurodegeneration. Some mutations found in ALS patients, overlooked because of their synonymous effect on the FUS protein, exert a deleterious effect via their missense consequence on the overlapping altFUS protein. Hence, both proteins, FUS and altFUS, are involved in the aetiology and pathological hallmarks of ALS.

Furthermore, we used size exclusion chromatography and affinity purification MS (AP-MS) to explore altFUS interactome, demonstrating its interaction with prohibitins, known regulators of autophagy. AP-MS in differentially SILAC-labelled cells highlighted protein-protein interaction differences between ALS-linked mutants and wild-type altFUS. Functional annotation revealed in altFUS mutants a loss of proteins involved in the stress response.

FUS dual-coding nature is not an exception. 56% of ALS-associated genes possess an altORF with experimental evidence in OpenProt. Re-analysis of ALS proteomic studies reveal altORFs as hidden players and potential biomarkers, emphasizing the importance of proteogenomic pipelines to better understand human diseases.

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A Consensus Proteomic Analysis of Alzheimer’s Disease Brain and Cerebrospinal Fluid Reveals Early Changes Associated with Microglia and Astrocyte Activation

Erik Johnson1; Eric Dammer1; Duc Duong1; Lingyan Ping1; Maotian Zhou1; Luming Yin1; Lenora Higginbotham1; Andrew Guajardo2; Bartholomew White2; Juan Troncoso2; Madhav Thambisetty3; Thomas Montine4; Edward Lee5; John Q. Trojanowski5; Thomas Beach6; Eric Reiman7; Vahram Haroutunian8; Minghui Wang8; Eric Schadt8; Bin Zhang8; Dennis Dickson9; Nilufer Taner9; Todd Golde10; Vladislav Petyuk11; Philip De Jager12; David Bennett13; Thomas Wingo1; Srikant Rangaraju1; Ihab Hajjar1; Joshua Shulman14; James Lah1; Allan Levey1; Nicholas Seyfried1
1Emory University, Atlanta, GA; 2Johns Hopkins School of Medicine, Baltimore, MD; 3National Institutes of Health, Bethesda, MD; 4Stanford University, Palo Alto, CA; 5University of Pennsylvania, Philadelphia, PA; 6Banner Sun Health Research Institute, Sun City, AZ; 7Banner Alzheimer's Institute, Phoenix, AZ; 8Mount Sinai School of Medicine, New York, NY; 9Mayo Clinic, Jacksonville, FL; 10University of Florida, Gainesville, FL; 11Pacific Northwest National Laboratory, Richland, WA; 12Columbia University, New York, NY; 13Rush University, Chicago, IL; 14Baylor College of Medicine, Houston, TX

Our understanding of the biological changes in the brain associated with Alzheimer’s disease (AD) pathology and cognitive impairment remains incomplete.  To increase our understanding of these changes, we analyzed dorsolateral prefrontal cortex of control, asymptomatic AD, and AD brains from four different centers by label-free quantitative mass spectrometry and weighted protein co-expression analysis to obtain a consensus protein co-expression network of AD brain.  This network consisted of 13 protein co-expression modules.  Six of these modules correlated with amyloid-β plaque burden, tau neurofibrillary tangle burden, cognitive function, and clinical functional status, and were altered in asymptomatic AD, AD, or in both disease states.  These six modules reflected synaptic, mitochondrial, sugar metabolism, extracellular matrix, cytoskeletal, and RNA binding/splicing biological functions.  The identified protein network modules were preserved in a community-based cohort analyzed by a different quantitative mass spectrometry approach.  They were also preserved in temporal lobe and precuneus brain regions.  Some of the modules were influenced by aging, and showed changes in other neurodegenerative diseases such as frontotemporal dementia and corticobasal degeneration.  The module most strongly associated with AD pathology and cognitive impairment was the sugar metabolism module.  This module was enriched in AD genetic risk factors, and was also highly enriched in microglia and astrocyte protein markers associated with an anti-inflammatory state, suggesting that the biological functions it represents serve a protective role in AD.  Proteins from the sugar metabolism module were increased in cerebrospinal fluid from asymptomatic AD and AD cases, highlighting their potential as biomarkers of the altered brain network.  In this study of >2000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brain that may serve as therapeutic targets and fluid biomarkers for the disease.

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