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The influence of APOEε4 on the pTau interactome in sporadic Alzheimer's disease

Thierry, Manon; Ponce, Jackeline; Martà-Ariza, Mitchell; Askenazi, Manor; Faustin, Arline; Leitner, Dominique; Pires, Geoffrey; Kanshin, Evgeny; Drummond, Eleanor; Ueberheide, Beatrix; Wisniewski, Thomas
APOEε4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). Although APOEε4 is known to promote Aβ pathology, recent data also support an effect of APOE polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across APOE genotypes in the frontal cortex of 10 advanced AD cases (n = 5 APOEε3/ε3 and n = 5 APOEε4/ε4), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-Aβ 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases (n = 11 APOEε3/ε3 and n = 10 APOEε4/ε4). Our dataset includes 1130 and 1330 proteins enriched in IPPHF1 samples from APOEε3/ε3 and APOEε4/ε4 groups (fold change ≥ 1.50, IPPHF1 vs IPIgG ctrl). We identified 80 and 68 proteins as probable pTau interactors in APOEε3/ε3 and APOEε4/ε4 groups, respectively (SAINT score ≥ 0.80; false discovery rate (FDR) ≤ 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOEε3/ε3 vs APOEε4/ε4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOEε4/ε4 vs APOEε3/ε3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOEε4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOEε4/ε4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to Aβ-affected brain regions in APOEε4 carriers, paving the way to the identification of new therapeutic targets.
PMID: 38772917
ISSN: 1432-0533
CID: 5654472

Spatial proteomics of hippocampal subfield-specific pathology in Alzheimer's disease and primary age-related tauopathy

Walker, Jamie M; Orr, Miranda E; Orr, Timothy C; Thorn, Emma L; Christie, Thomas D; Yokoda, Raquel T; Vij, Meenakshi; Ehrenberg, Alexander J; Marx, Gabriel A; McKenzie, Andrew T; Kauffman, Justin; Selmanovic, Enna; Wisniewski, Thomas; Drummond, Eleanor; White, Charles L; Crary, John F; Farrell, Kurt; Kautz, Tiffany F; Daoud, Elena V; Richardson, Timothy E
INTRODUCTION/BACKGROUND:Alzheimer's disease (AD) and primary age-related tauopathy (PART) both harbor 3R/4R hyperphosphorylated-tau (p-tau)-positive neurofibrillary tangles (NFTs) but differ in the spatial p-tau development in the hippocampus. METHODS:Using Nanostring GeoMx Digital Spatial Profiling, we compared protein expression within hippocampal subregions in NFT-bearing and non-NFT-bearing neurons in AD (n = 7) and PART (n = 7) subjects. RESULTS:Proteomic measures of synaptic health were inversely correlated with the subregional p-tau burden in AD and PART, and there were numerous differences in proteins involved in proteostasis, amyloid beta (Aβ) processing, inflammation, microglia, oxidative stress, and neuronal/synaptic health between AD and PART and between definite PART and possible PART. DISCUSSION/CONCLUSIONS:These results suggest subfield-specific proteome differences that may explain some of the differences in Aβ and p-tau distribution and apparent pathogenicity. In addition, hippocampal neurons in possible PART may have more in common with AD than with definite PART, highlighting the importance of Aβ in the pathologic process. HIGHLIGHTS/CONCLUSIONS:Synaptic health is inversely correlated with local p-tau burden. The proteome of NFT- and non-NFT-bearing neurons is influenced by the presence of Aβ in the hippocampus. Neurons in possible PART cases share more proteomic similarities with neurons in ADNC than they do with neurons in definite PART cases.
PMID: 37777848
ISSN: 1552-5279
CID: 5633692

Similar brain proteomic signatures in Alzheimer's disease and epilepsy

Leitner, Dominique; Pires, Geoffrey; Kavanagh, Tomas; Kanshin, Evgeny; Askenazi, Manor; Ueberheide, Beatrix; Devinsky, Orrin; Wisniewski, Thomas; Drummond, Eleanor
The prevalence of epilepsy is increased among Alzheimer's Disease (AD) patients and cognitive impairment is common among people with epilepsy. Epilepsy and AD are linked but the shared pathophysiological changes remain poorly defined. We aim to identify protein differences associated with epilepsy and AD using published proteomics datasets. We observed a highly significant overlap in protein differences in epilepsy and AD: 89% (689/777) of proteins altered in the hippocampus of epilepsy patients were significantly altered in advanced AD. Of the proteins altered in both epilepsy and AD, 340 were altered in the same direction, while 216 proteins were altered in the opposite direction. Synapse and mitochondrial proteins were markedly decreased in epilepsy and AD, suggesting common disease mechanisms. In contrast, ribosome proteins were increased in epilepsy but decreased in AD. Notably, many of the proteins altered in epilepsy interact with tau or are regulated by tau expression. This suggests that tau likely mediates common protein changes in epilepsy and AD. Immunohistochemistry for Aβ and multiple phosphorylated tau species (pTau396/404, pTau217, pTau231) showed a trend for increased intraneuronal pTau217 and pTau231 but no phosphorylated tau aggregates or amyloid plaques in epilepsy hippocampal sections. Our results provide insights into common mechanisms in epilepsy and AD and highlights the potential role of tau in mediating common pathological protein changes in epilepsy and AD.
PMID: 38289539
ISSN: 1432-0533
CID: 5627492

Compilation of reported protein changes in the brain in Alzheimer's disease

Askenazi, Manor; Kavanagh, Tomas; Pires, Geoffrey; Ueberheide, Beatrix; Wisniewski, Thomas; Drummond, Eleanor
Proteomic studies of human Alzheimer's disease brain tissue have potential to identify protein changes that drive disease, and to identify new drug targets. Here, we analyse 38 published Alzheimer's disease proteomic studies, generating a map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimer's disease, mild cognitive impairment, advanced Alzheimer's disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a searchable database (NeuroPro). We found 848 proteins were consistently altered in 5 or more studies. Comparison of protein changes in early-stage and advanced Alzheimer's disease revealed proteins associated with synapse, vesicle, and lysosomal pathways show change early in disease, but widespread changes in mitochondrial associated protein expression change are only seen in advanced Alzheimer's disease. Protein changes were similar for brain regions considered vulnerable and regions considered resistant. This resource provides insight into Alzheimer's disease brain protein changes and highlights proteins of interest for further study.
PMID: 37491476
ISSN: 2041-1723
CID: 5592142

Use of Affinity Purification-Mass Spectrometry to Identify Phosphorylated Tau Interactors in Alzheimer's Disease

Pires, Geoffrey; Ueberheide, Beatrix; Wisniewski, Thomas; Drummond, Eleanor
Phosphorylated tau is the main protein present in neurofibrillary tangles, the presence of which is a key neuropathological hallmark of Alzheimer's disease (AD). The toxic effects of phosphorylated tau are likely mediated by interacting proteins; however, methods to identify these interacting proteins comprehensively in human brain tissue are limited. Here, we describe a method that enables the efficient identification of hundreds of proteins that interact with phosphorylated tau (pTau), using affinity purification-mass spectrometry (AP-MS) on human, fresh-frozen brain tissue from donors with AD. Tissue is homogenized using a gentle technique that preserves protein-protein interactions, and co-immunoprecipitation of pTau and its interacting proteins is performed using the PHF1 antibody. The resulting protein interactors are then identified using label-free quantitative liquid chromatography-mass spectrometry (LC-MS)/MS. The Significance Analysis of INTeractome (SAINT) algorithm is used to determine which proteins significantly interact with pTau. This approach enables the detection of an abundance of all 6 isoforms of tau, 23 phosphorylated residues on tau, and 125 significant pTau protein interactors, in human AD brain tissue.
PMID: 36399275
ISSN: 1940-6029
CID: 5371722

The amyloid plaque proteome in early onset Alzheimer's disease and Down syndrome

Drummond, Eleanor; Kavanagh, Tomas; Pires, Geoffrey; Marta-Ariza, Mitchell; Kanshin, Evgeny; Nayak, Shruti; Faustin, Arline; Berdah, Valentin; Ueberheide, Beatrix; Wisniewski, Thomas
Amyloid plaques contain many proteins in addition to beta amyloid (Aβ). Previous studies examining plaque-associated proteins have shown these additional proteins are important; they provide insight into the factors that drive amyloid plaque development and are potential biomarkers or therapeutic targets for Alzheimer's disease (AD). The aim of this study was to comprehensively identify proteins that are enriched in amyloid plaques using unbiased proteomics in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. We focused our study on early onset AD as the drivers of the more aggressive pathology development in these cases is unknown and it is unclear whether amyloid-plaque enriched proteins differ between subtypes of early onset AD. Amyloid plaques and neighbouring non-plaque tissue were microdissected from human brain sections using laser capture microdissection and label-free LC-MS was used to quantify the proteins present. 48 proteins were consistently enriched in amyloid plaques in EOAD and DS. Many of these proteins were more significantly enriched in amyloid plaques than Aβ. The most enriched proteins in amyloid plaques in both EOAD and DS were: COL25A1, SMOC1, MDK, NTN1, OLFML3 and HTRA1. Endosomal/lysosomal proteins were particularly highly enriched in amyloid plaques. Fluorescent immunohistochemistry was used to validate the enrichment of four proteins in amyloid plaques (moesin, ezrin, ARL8B and SMOC1) and to compare the amount of total Aβ, Aβ40, Aβ42, phosphorylated Aβ, pyroglutamate Aβ species and oligomeric species in EOAD and DS. These studies showed that phosphorylated Aβ, pyroglutamate Aβ species and SMOC1 were significantly higher in DS plaques, while oligomers were significantly higher in EOAD. Overall, we observed that amyloid plaques in EOAD and DS largely contained the same proteins, however the amount of enrichment of some proteins was different in EOAD and DS. Our study highlights the significant enrichment of many proteins in amyloid plaques, many of which may be potential therapeutic targets and/or biomarkers for AD.
PMID: 35418158
ISSN: 2051-5960
CID: 5201962

It takes more than tau to tangle: using proteomics to determine how phosphorylated tau mediates toxicity in neurodegenerative diseases

Pires, Geoffrey; Drummond, Eleanor
PMID: 33818497
ISSN: 1673-5374
CID: 4855602

Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients

Leitner, Dominique F; Mills, James D; Pires, Geoffrey; Faustin, Arline; Drummond, Eleanor; Kanshin, Evgeny; Nayak, Shruti; Askenazi, Manor; Verducci, Chloe; Chen, Bei Jun; Janitz, Michael; Anink, Jasper J; Baayen, Johannes C; Idema, Sander; van Vliet, Erwin A; Devore, Sasha; Friedman, Daniel; Diehl, Beate; Scott, Catherine; Thijs, Roland; Wisniewski, Thomas; Ueberheide, Beatrix; Thom, Maria; Aronica, Eleonora; Devinsky, Orrin
OBJECTIVE:To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to epilepsy control patients. METHODS:For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected post-mortem brain tissue of 12 SUDEP and 14 non-SUDEP epilepsy patients. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from mesial temporal lobe epilepsy (MTLE) patients: 6 low-risk and 8 high-risk SUDEP as determined by a short (< 50 seconds) or prolonged (≥ 50 seconds) postictal generalized EEG suppression (PGES) that may indicate severely depressed brain activity impairing respiration, arousal, and protective reflexes. RESULTS:In autopsy hippocampus and cortex, we observed no proteomic differences between SUDEP and non-SUDEP epilepsy patients, contrasting with our previously reported robust differences between epilepsy and non-epilepsy control patients. Transcriptomics in hippocampus and cortex from surgical epilepsy patients segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 lncRNAs, three pending) in hippocampus. CONCLUSION/CONCLUSIONS:The SUDEP proteome and high-risk SUDEP transcriptome were similar to other epilepsy patients in hippocampus and frontal cortex, consistent with diverse epilepsy syndromes and comorbidities associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions may identify molecular mechanisms of SUDEP.
PMID: 33910938
ISSN: 1526-632x
CID: 4852152

Proteomic differences in the hippocampus and cortex of epilepsy brain tissue

Pires, Geoffrey; Leitner, Dominique; Drummond, Eleanor; Kanshin, Evgeny; Nayak, Shruti; Askenazi, Manor; Faustin, Arline; Friedman, Daniel; Debure, Ludovic; Ueberheide, Beatrix; Wisniewski, Thomas; Devinsky, Orrin
Epilepsy is a common neurological disorder affecting over 70 million people worldwide, with a high rate of pharmaco-resistance, diverse comorbidities including progressive cognitive and behavioural disorders, and increased mortality from direct (e.g. sudden unexpected death in epilepsy, accidents, drowning) or indirect effects of seizures and therapies. Extensive research with animal models and human studies provides limited insights into the mechanisms underlying seizures and epileptogenesis, and these have not translated into significant reductions in pharmaco-resistance, morbidities or mortality. To help define changes in molecular signalling networks associated with seizures in epilepsy with a broad range of aetiologies, we examined the proteome of brain samples from epilepsy and control cases. Label-free quantitative mass spectrometry was performed on the hippocampal cornu ammonis 1-3 region (CA1-3), frontal cortex and dentate gyrus microdissected from epilepsy and control cases (n = 14/group). Epilepsy cases had significant differences in the expression of 777 proteins in the hippocampal CA1 - 3 region, 296 proteins in the frontal cortex and 49 proteins in the dentate gyrus in comparison to control cases. Network analysis showed that proteins involved in protein synthesis, mitochondrial function, G-protein signalling and synaptic plasticity were particularly altered in epilepsy. While protein differences were most pronounced in the hippocampus, similar changes were observed in other brain regions indicating broad proteomic abnormalities in epilepsy. Among the most significantly altered proteins, G-protein subunit beta 1 (GNB1) was one of the most significantly decreased proteins in epilepsy in all regions studied, highlighting the importance of G-protein subunit signalling and G-protein-coupled receptors in epilepsy. Our results provide insights into common molecular mechanisms underlying epilepsy across various aetiologies, which may allow for novel targeted therapeutic strategies.
PMID: 34159317
ISSN: 2632-1297
CID: 5387022

Phosphorylated tau interactome in the human Alzheimer's disease brain

Drummond, Eleanor; Pires, Geoffrey; MacMurray, Claire; Askenazi, Manor; Nayak, Shruti; Bourdon, Marie; Safar, Jiri; Ueberheide, Beatrix; Wisniewski, Thomas
Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer's disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer's disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer's disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer's disease.
PMID: 32812023
ISSN: 1460-2156
CID: 4566892