Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Mapping of proteoforms, a method that enables massively parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. We applied Iterative Mapping to tau, a key protein in neurodegenerative diseases, using 12 site-specific antibodies. The tau proteoform assay demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median coefficient of variation <5.5%) and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. Analysis of relevant biological samples, including organoids, mouse brains and human Alzheimer’s disease samples, revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The nonrandom distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Iterative Mapping provides insights into proteoform complexity at the single-molecule level, with implications for understanding protein regulation in neurodegenerative diseases and beyond. Iterative Mapping of proteoforms enables large-scale measurement of intact proteoforms with single-molecule resolution. The approach is demonstrated here by quantifying tau proteoform groups across control samples of known composition, model systems used in tauopathy research, and human-derived brain tissue samples.
James Joly, V. Budamagunta, Zheng-Jian Zhang et al.· Nature Methods· 1 citation
Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-tau), currently lack effective therapies and can occur sporadically or be inherited when associated with MAPT gene mutations. Exon 10 and adjacent introns of the MAPT gene are a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression and 4R-specific gain-of-function mutations that generate aggregation-prone tau. For these 4R tauopathies, a targeted messenger RNA (mRNA) splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. We have developed splicing modulator compounds (SMCs) that promote MAPT exon 10 exclusion and demonstrated their efficacy in neurons derived from patients with FTD carrying the tau Pro301→Leu (P301L) gain-of-function mutation or the tau Ser305→Asn (S305N) splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau) and oligomeric and insoluble tau proteoforms, thereby rescuing tau-associated neuronal toxicity. A lead SMC corrected the 3R/4R splice ratio in vivo and reduced pTau in the brain of a human gene-replacement mouse model expressing the tau Asn279→Lys (N279K) splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish MAPT pre-mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies.
M. Silva, Hannah Lindmeier, Paolo Pigini et al.· Science Translational Medici...· 1 citation
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