Findings establish spatial proteoforms as essential units of pathogenic analysis and reveal disease-relevant signals invisible to bulk tissue approaches in Alzheimer's disease.
Abstract
Alzheimer’s disease (AD) pathogenesis involves complex, multifactorial changes to the brain proteome that conventional unfractionated analyses may obscure. Proteins frequently occupy multiple subcellular compartments as spatial proteoforms, yet the contribution of aberrant protein localization to AD pathogenesis remains poorly understood. To address this, we fractionated post-mortem human hippocampi from 13 AD and 14 non-AD individuals into four subcellular fractions and quantified 6,123 proteins by TMT-LC-MS. Although 75% of proteins were detected in more than one fraction, 78% of significant AD-associated alterations were restricted to a single fraction, demonstrating that subcellular localization is a primary determinant of disease vulnerability. Discordant abundance patterns between fractions revealed retromer complex mislocalization, nuclear transport dysfunction, and insoluble protein accumulation, with the endosomal-lysosomal and protein folding pathways most consistently perturbed. To examine how these perturbations evolve with disease progression, we applied the QUAD strategy to measure protein degradation in two fractions of APPswePS1delta9 mouse cortex at 2, 5, and 12 months. Degradation rates diverged between fractions and genotypes in an age-dependent manner, and cross-dataset comparison identified six proteins altered at the earliest pre-pathological timepoint, implicating vesicle transport and proteostasis disruption as initiating features of AD. These findings establish spatial proteoforms as essential units of pathogenic analysis and reveal disease-relevant signals invisible to bulk tissue approaches.
This dissertation explores the hypothesis that G4-forming nucleic acids represent a mechanistic link between cellular stress, RNA biology, and tau aggregation in AD and establishes G-quadruplexes as previously underappreciated regulators of tau biology and implicates RNA structure as an active contributor to AD pathogenesis.
Proteome-wide analyses of human tissue have transformed our understanding of disease, but provide limited insight into protein localisation, a functionally informative dimension of the proteome. In Alzheimer’s disease, amyloid-β and tau exhibit aberrant localisation, yet whether spatial reorganisation extends proteome-wide has remained inaccessible to abundance-based proteomics. Here, we develop comparative subcellular proteomics applied to dorsolateral prefrontal cortex from 75 individuals spanning the Alzheimer’s disease-resilience spectrum, modelling protein localisation across disease. We identify 217 disease-associated localisation shifts enriched for endolysosomal function, intracellular trafficking, and RNA processing, and resolve tau proteoforms within insoluble aggregates. Our strongest localisation candidates show only modest differences in whole-tissue abundance, highlighting disease biology inaccessible to conventional proteomics. We validate co-localisation of CSNK1A1 with pathological tau and identify an unexpected neuronal localisation pattern for SCAI, a cancer-associated protein not previously characterised in human brain, highlighting the discovery potential of subcellular proteomics in tissue.
Helen A. Jolly, Paula Seghers, Kaleah Balcomb et al.· bioRxiv· 0 citations
Alzheimer’s disease (AD) is defined by amyloid-β (Aβ) plaques and tau tangles, yet the inflammation that comes with it is only partly localized where those lesions accumulate. Using spatial transcriptomics on 16 human hippocampal sections containing adjacent cortex, we found that plaques concentrated in grey matter, especially cortex. In contrast, the strongest inflammatory response occupied white matter and increased with distance from Aβ-positive spots. This inflammatory signature increased with Braak stage in an independent 31-subject hippocampal bulk proteomic cohort. The white-matter environment revealed a distinct chemistry, with lipidomics showing cortical white matter gaining cholesteryl esters, lysosomal storage lipids and peroxidation-prone polyunsaturated species while losing myelin lipids, alongside carbonyl, glycation and iron-handling signatures. In an external single-nucleus cohort, an oligodendrocyte lipid-droplet program tracked cognitive decline after adjustment for amyloid and tangles, and the same reactive-glia chemistry recurred above expression-matched nulls across seven neurodegenerative datasets. We propose that this lipid-rich glial environment is a metastable, primed state, and that Aβ and tau act as catalysts that tip it toward a self-sustaining inflammatory reaction. Highlights Plaques concentrate in grey matter, whereas inflammatory programs increase with distance and peak in glial/myelin-rich white matter. The white-matter response strengthens with Braak stage and is characterized by carbonyl stress, lipid storage, and myelin loss. An oligodendrocyte lipid-droplet program tracks cognitive decline after adjustment for Aβ and tau. A lipid-droplet reactive-glial state recurs across neurodegenerative diseases with distinct triggers and vulnerable cells. Aβ and pTau may tip a metastable tissue environment already primed by lipid and carbonyl stress. Graphical abstract
Aurélien M. Badina, Eléonore Raveloson, I. Meister et al.· bioRxiv· 0 citations
Alzheimer’s disease (AD) is a progressive and complex neurodegenerative disorder characterized by amyloid-β (Aβ) plaques and hyperphosphorylated tau, accompanied by changes in synaptic function, metabolism, proteostasis, neuroinflammation, glial activity, and extracellular signaling. The interaction between these processes contributes to the molecular heterogeneity observed between individuals, disease stages, and clinical subtypes. In this thesis, I used proteomics as a central approach to investigate AD from complementary perspectives, including disease heterogeneity, extracellular vesicle (EV) signaling, therapeutic intervention, and I developed an accessible proteomics workflow. I first investigated molecular differences between early-onset familial AD (EOAD) and late-onset AD (LOAD) using 115 human post-mortem brain tissues. EOAD showed stronger dysregulation of mitochondrial oxidative phosphorylation and synaptic pathways, while peptide-level analysis identified phosphorylated tau isoforms as candidate subtype- and stage-specific biomarkers. In addition, Aβ positivity in cognitively normal individuals was compatible with both preclinical AD and resilience, highlighting a biologically active prodromal phase. I next investigated the relationship between extracellular matrix remodeling and EV composition in the APP/PSEN1 mouse model. In the 5xFAD model, treatment with the neurotrophin mimetic BNN27 altered proteins and pathways related to Aβ metabolism, synaptic function, and neuroinflammation, accompanied by reduced amyloid plaque burden, increased hippocampal neurogenesis, and improved memory performance. Finally, I developed a cost-effective and reproducible sTRAP workflow that improved protein and peptide identification from low-input samples and captured disease-relevant proteomic signatures in 5xFAD hippocampal tissue. Altogether, these findings demonstrate the potential of proteomics to characterize the molecular complexity of AD and reveal interconnected changes across different levels of disease biology. They further support a view of AD as a progressive disruption of cellular and intercellular communication, emphasizing the need for multi-targeted approaches to understand and treat the disease.
Visual dysfunction is increasingly recognized as an important feature of Alzheimer’s disease, and substantial retinal changes have been documented across multiple studies. Yet the molecular changes underlying retinal neurodegeneration and which retinal protein signatures best track cerebral pathology remain incompletely defined. Here, we performed comprehensive mass spectrometry-based proteomics on paired retinal and hippocampal tissue from the same postmortem donors (8 AD, 8 non-demented controls) to identify disease-associated molecular signatures and assess their overlap between these tissues. Using a sequential dual-extraction protocol, we identified 372 differentially abundant retinal proteins in AD, including established APP-processing regulators (SORL1, BACE1) and synaptic proteins. Retinal proteomes clearly separated AD from controls in principal component analysis, indicating robust AD-related molecular differences in the retina. Notably, 87% of proteins were detected in both retina and hippocampus, with 64 differentially abundant proteins shared between tissues, some of which showed strong cross-tissue correlation. Several retinal proteins also correlated with neuropathological disease stage. Functional enrichment analysis revealed convergent alterations in synaptic organization, cytoskeletal dynamics, mitochondrial function, cell adhesion, and APP metabolism in both tissues. Cell-type mapping using single-cell retinal reference data indicated that most proteomic changes were broadly distributed across cell types, though some proteins showed enrichment in specific populations, such as SORL1 in microglia and EYS in photoreceptors. The molecular changes identified here offer a potential basis for the retinal alterations previously documented through in vivo imaging and histological studies. Their similarities with brain pathology further support the retina as a promising window for assessing cerebral disease.
Routine and accessible plasma measures can be leveraged to recover reproducible, biologically distinct progression modules that improve characterization of heterogeneous AD and have practical value for risk stratification, trial enrichment, or treatment monitoring.
R. R. Butler, M. Brown, A. Weber et al.· medRxiv· 0 citations
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