Integrated Proteomics Reveals Brain-Based Cerebrospinal Fluid Biomarkers in Asymptomatic and Symptomatic Alzheimer’s Disease
Open accessPreprint — published as 10.1126/sciadv.aaz9360 · Paper page
Abstract
Abstract Alzheimer’s disease (AD) features a complex web of pathological processes beyond amyloid accumulation and tau-mediated neuronal death. To meaningfully advance AD therapeutics, there is an urgent need for novel biomarkers that comprehensively reflect these disease mechanisms. Here we applied an integrative proteomics approach to identify cerebrospinal fluid (CSF) biomarkers linked to a diverse set of pathophysiological processes in the diseased brain. Using multiplex proteomics, we identified >3,500 proteins across 40 CSF samples from control and AD patients and >12,000 proteins across 48 postmortem brain tissues from control, asymptomatic AD (AsymAD), AD, and other neurodegenerative cases. Co-expression network analysis of the brain tissues resolved 44 protein modules, nearly half of which significantly correlated with AD neuropathology. Fifteen modules robustly overlapped with proteins quantified in the CSF, including 271 CSF markers highly altered in AD. These 15 overlapping modules were collapsed into five panels of brain-linked fluid markers representing a variety of cortical functions. Neuron-enriched synaptic and metabolic panels demonstrated decreased levels in the AD brain but increased levels in diseased CSF. Conversely, glial-enriched myelination and immunity panels were highly increased in both the brain and CSF. Using high-throughput proteomic analysis, proteins from these panels were validated in an independent CSF cohort of control, AsymAD, and AD samples. Remarkably, several validated markers were significantly altered in AsymAD CSF and appeared to stratify subpopulations within this cohort. Overall, these brain-linked CSF biomarker panels represent a promising step toward a physiologically comprehensive tool that could meaningfully enhance the prognostic and therapeutic management of AD.
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OLFML3, SPON1, and SLIT2 stood out in this study and reference study 1 (data vi).
“OLFML3, SPON1, and SLIT2 stood out in this study and reference study 1 (data vi) [33].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
The figure listing brain-linked CSF panels includes OLFML3 in the vascular panel and SPON1 in the glial-immunity panel (Figure S2 section; page unavailable). Our copy does not identify SLIT2 or establish that all three proteins stood out, so it supports only part of the attributed claim. (LLM, conf=0.78)
Experimental — accuracy not yet measuredSMOC1 and GPNMB have been reported as putative CSF biomarkers in previous studies.
“While, SMOC1 and GPNMB have been reported as putative CSF biomarkers in previous studies [18, 33, 62]”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
The replication-cohort section identifies SMOC1 as a validated brain-linked CSF marker, supporting that part of the claim. A search of our copy for GPNMB found no passage reporting it as a CSF biomarker, so this source does not support the GPNMB part. (LLM, conf=0.88)
Experimental — accuracy not yet measuredMitochondrial changes can co-occur with amyloid deposition early in the brain of asymptomatic cases with amyloid pathology, as well as mild cognitive impairment subjects.
“Mitochondrial changes can co-occur with amyloid deposition early in the brain of asymptomatic cases with amyloid pathology, as well as mild cognitive impairment subjects [18, 33, 75].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
The section on asymptomatic AD brain proteomics (page unavailable) reports high amyloid burden and presymptomatic protein changes in cognitively normal cases. The brain-network analysis links a mitochondrial protein module to AD pathology, but does not establish mitochondrial changes in the asymptomatic cases specifically. The paper's brain cohorts do not include a mild cognitive impairment group, so that part of the claim is unsupported. (LLM, conf=0.85)
Experimental — accuracy not yet measuredMitochondrial protein decreases in Alzheimer's disease CSF and serum have rarely been reported until the availability of deep CSF/serum profiling.
“In this study, we identified consistent and evident mitochondrial protein decreases in AD CSF and serum samples, which have rarely reported until the availability of the deep CSF/serum profiling (our reference datasets) [25, 33].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
The ‘CSF Proteome Reveals Markers Significantly Altered in AD’ section describes deep CSF protein profiling, supporting that part of the citation. The paper analyzes brain and CSF datasets, not serum, and the passages do not say that mitochondrial-protein decreases in AD CSF or serum had rarely been reported; its Discussion instead describes predominantly increased abundance of neuronal metabolic-panel proteins in AD CSF. Page numbers were not available in our copy. (LLM, conf=0.80)
Experimental — accuracy not yet measuredSeveral other groups used similar strategies in AD biomarker studies.
“In addition, several other groups used the similar strategies in AD biomarker studies [33, 34].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
The 5 reference datasets were mined from 2 independent biofluid proteome studies of AD.
“The 5 reference datasets were mined from 2 independent biofluid proteome studies of AD [33, 34].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
In this study, consistent and evident mitochondrial protein decreases were identified in Alzheimer's disease CSF and serum samples.
“In this study, we identified consistent and evident mitochondrial protein decreases in AD CSF and serum samples, which have rarely reported until the availability of the deep CSF/serum profiling (our reference datasets) [25, 33].”
From Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease (2020) · cites this paper as [33]
Recent studies have identified additional potential biomarkers from M4 and other brain modules by deep mass spectrometry-based discovery on fractionated cerebrospinal fluid.
“Indeed, recent studies have identified a number of additional potential biomarkers from M4 and other brain modules by deep mass spectrometry-based discovery on fractionated CSF35,36,54.”
Targets among overlapping modules show highly discordant changes at the protein and RNA levels.
“While differential protein expression within these overlapping modules is reasonably concordant (R~0.5), we have repeatedly observed targets among these modules with highly discordant changes at the protein and RNA levels [62–64].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Differential protein expression within overlapping modules is reasonably concordant (R~0.5).
“While differential protein expression within these overlapping modules is reasonably concordant (R~0.5), we have repeatedly observed targets among these modules with highly discordant changes at the protein and RNA levels [62–64].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Nearly a dozen network-based analyses of the AD proteome have been performed in the human brain, including the dorsolateral prefrontal cortex, temporal cortex, and other cortical regions heavily affected in AD.
“Nearly a dozen informative network-based analyses of the AD proteome have been performed in the human brain [62–64, 74, 78, 96, 104–108], including the dorsolateral prefrontal cortex (DLPFC), temporal cortex, and other cortical regions heavily affected in AD.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Only 30-40% of modules in the AD brain network proteome overlap with those of the network transcriptome.
“Only 30–40% of modules in the AD brain network proteome overlap with those of the network transcriptome [62, 63].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
The highly conserved microglial/astrocytic module features hub proteins implicated in neuroprotection, such as moesin.
“This highly conserved module features hub proteins implicated in neuroprotection, such as the membrane receptor-cytoskeleton cross-linking protein moesin (MSN) [62–64, 96].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Multiple network proteomes have resolved a large module enriched with microglial and astrocytic proteins that demonstrates progressively increasing abundance throughout AsymAD and AD.
“Multiple network proteomes have resolved a large module enriched with microglial and astrocytic proteins that demonstrates progressively increasing abundance levels throughout AsymAD and AD.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
In our unbiased network analysis of >8000 proteins from AD DLPFC, we resolved an ECM-associated module with highly significant endothelial enrichment and relatively weak glial co-expression.
“Among the resulting 44 modules, we resolved an ECM-associated module with highly significant endothelial enrichment and relatively weak glial co-expression.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
The AD brain network proteome features conserved upregulation of oligodendrocyte-enriched modules associated with myelination.
“The AD brain network proteome also features conserved upregulation of oligodendrocyte-enriched modules associated with myelination (Fig. 1).”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Proteomic network analyses have demonstrated modest upregulation of a highly conserved oligodendrocyte-enriched myelination module in both asymptomatic and symptomatic disease.
“proteomic network analyses of the AD brain have demonstrated modest upregulation of a highly conserved oligodendrocyte-enriched myelination module in both asymptomatic and symptomatic disease [62–64, 74, 96].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Modules enriched with RNA binding and splicing proteins are consistently upregulated in the AD network proteome.
“Modules enriched with RNA binding and splicing proteins are also consistently upregulated in the AD network proteome (Fig. 1).”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
One other highly conserved AD-associated module is consistently downregulated throughout AD and strongly associated with the cytoskeleton.
“network proteomics has revealed one other highly conserved AD-associated module with possible links to this phase of disease. Unlike the RNA-binding/splicing modules, this module is consistently downregulated throughout AD and strongly associated with the cytoskeleton (Fig. 1) [62, 63, 96].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
CSF features stark elevations in both synaptic and metabolic proteins in the asymptomatic stages of disease.
“CSF, which features stark elevations in both types of proteins in the asymptomatic stages of disease [63].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Emerging CSF proteomic data has revealed alterations in a number of synapse-associated proteins in preclinical disease.
“emerging cerebrospinal fluid (CSF) proteomic data has revealed alterations in a number of synapse-associated proteins in preclinical disease [63].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
The results of these validation studies were used to narrow down these biomarker panels from roughly 300 to 60 proteins across the five panels.
“The results of these validation studies were then used to narrow down these biomarker panels from roughly 300 to 60 proteins across the five panels.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
These ~300 CSF AD targets were segregated into five systems-based biomarker panels representing a wide range of brain pathophysiology, including synaptic transmission, vascular biology, myelination, glial-mediated inflammation, and energy metabolism.
“Based on their corresponding brain modules, these ~300 CSF AD targets were then segregated into five systems-based biomarker panels representing a wide range of brain pathophysiology, including synaptic transmission, vascular biology, myelination, glial-mediated inflammation, and energy metabolism.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Proteins from these five panels were validated in multiple additional CSF cohorts, totaling >500 spinal fluid samples.
“Using high-throughput TMT proteomic analysis, proteins from these five panels were validated in multiple additional CSF cohorts, totaling >500 spinal fluid samples.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Higginbotham et al. used an integrative proteomic approach to examine the statistical overlap of the AD brain network proteome (DLPFC) with differential expression in the AD CSF proteome.
“In another recent large-scale study, Higginbotham et al. used an integrative proteomic approach to examine the statistical overlap of the AD brain network proteome (DLPFC) with differential expression in the AD CSF proteome [63].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
These 15 brain modules were high-yield sources of markers differentially expressed in AD CSF, collectively harboring nearly 300 proteins with significantly altered levels in AD spinal fluid compared to controls.
“These 15 brain modules were also high-yield sources of markers differentially expressed in AD CSF, collectively harboring nearly 300 proteins with significantly altered levels in AD spinal fluid compared to controls.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
The final CSF marker panels demonstrated AD-specificity and altered levels in AsymAD spinal fluid, indicating a potential role for these panels in preclinical disease stages.
“Interestingly, the final CSF marker panels demonstrated AD-specificity and altered levels in AsymAD spinal fluid, indicating a potential role for these panels in preclinical disease stages.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
Fifteen of the 44 brain modules identified in this study strongly overlapped with the CSF proteome.
“Fifteen of the 44 brain modules identified in this study strongly overlapped with the CSF proteome.”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [63]
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References 122 · Cited by 367
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- Authors
- Higginbotham, L., Ping, L., Dammer, E. B., Duong, D. M., Zhou, M., Gearing, M., Johnson, E. C. B., Hajjar, I., Lah, J. J., Levey, A. I., & Seyfried, N. T.
- Affiliations
- Emory University (US)
- Corresponding author
- Levey, Allan I.
- Seyfried, Nicholas T.
- ORCID
- Venue
- Science Advances
- Publisher
- openRxiv
- Published
- 2019-10-16
- Volume
- 6
- Issue
- 43
- ISSN
- 2375-2548 (ISSN-L)
- PMC
- PMC7577712
- OpenAlex
- W3093903285
- Cited by
- 367
- References
- 122
- Citations by year
- 2026: 37 · 2025: 77 · 2024: 71 · 2023: 84 · 2022: 50 · 2021: 29 · 2020: 16 · 2019: 1 · 2012: 1
- FWCI
- 27.21
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by-nc
- Locations
- Science Advances (publishedVersion, cc-by-nc)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- Foundation for the National Institutes of Health
- Michael J. Fox Foundation for Parkinson's Research
- Alzheimer's Association
- Weston Brain Institute (11060)
- Alzheimer’s Research UK
- National Institute on Aging (RF1AG057470, R01AG057911, U01AG061357, U01AG046161, RF1AG057471, P50AG025688, R01AG057339, R01AG053960, R01AG061800)
- National Institute of Neurological Disorders and Stroke (P30NS055077)
- Topics
- Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
- Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- Alzheimer's disease, cerebrospinal fluid biomarkers, brain proteome, protein panels, network analysis, synaptic proteins, metabolic proteins, glial proteins, myelination, neuroinflammation, asymptomatic Alzheimer's disease
- MeSH terms
- Alzheimer Disease, Brain, Humans, Biomarkers, Proteome, Proteomics
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