Verani, M., Martufi, P., Petricca, L., Caricasole, A., Spiezia, M. C., Larsson, O., Tomei, L., Fodale, V., Munoz-Sanjuan, I., Vogt, T. F., Dominguez, C., Doherty, E. M., Lee, R., Cariulo, C. (2025). bioRxiv 2025.07.18.665500; https://doi.org/10.1101/2025.07.18.665500
Huntington’s disease (HD) is a progressive neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin (HTT) gene, resulting in a huntingtin protein with an expanded polyglutamine (polyQ) tract. Phosphorylation at residue S421 (pS421) affects HTT biology, including HTT stability, clearance, subcellular localization, toxicity, and axonal transport. Detection and quantification of S421-HTT phosphorylation in biological contexts have been challenging. This article reports a novel ultrasensitive immunoassay enabling detection of pS421-HTT. Using this assay, longitudinal assessment was conducted in HD mouse model tissues and detection in nonhuman primate cerebrospinal fluid (CSF) was achieved. PRKACA was identified as a kinase regulating endogenous pS421 in human cells. These assays and reagents will advance investigation into the biological relevance of pS421 in the natural history of HD.
Introduction
- Huntington’s disease (HD): caused by CAG expansion in HTT gene → mutant protein (mHTT) with polyQ repeat.
- HD is associated with misfolding and accumulation of pathogenic proteins; post-translational modifications (PTM) like phosphorylation play key roles.
- Therapeutic focus: lower HTT levels in the CNS; accurate quantification in models and human subjects is critical.
- pS421-HTT implicated in protein level modulation, subcellular localization, axonal transport, and toxicity.
- No quantitative and sensitive methods for pS421-HTT detection for robust preclinical profiling.
- Previous work: ultrasensitive immunoassays using SMC technology for other HTT PTMs.
Results
Development of an SMC assay for pS421-HTT
- Developed a sensitive SMC assay using MW1 and Ab2174/7D9 antibody pairs.
- Validated specificity by western blotting and SMC in cell and recombinant contexts.
- Evaluated pharmacological modulation using dopamine D2 receptor agonists/antagonists in HEK293T cells.
- Developed novel monoclonal antibody (mAb 7D9) with improved specificity for immunoassay optimization.
- Validated using recombinant protein and cell lysates; introduced 2B7 companion antibody to minimize bias.
Endogenous pS421-HTT detection
- Assay specificity confirmed via immunoprecipitation in HEK293T cells and HD mouse model tissues.
- Showed robust decreases in pS421-HTT signal following depletion, confirming assay specificity.
HTT-S421 phosphorylation in CNS tissues in HD mice
- Quantified pS421-HTT levels in Q7/Q7 (WT) and Q7/Q175 (HD) mice at various ages.
- Detected significant hypophosphorylation in HD mouse CNS tissue (cortex, hippocampus, cerebellum, striatum) with disease progression.
- No hypophosphorylation in heart tissue; decrease correlated with neurodegeneration, especially in striatum and hippocampus.
PRKACA kinase modulation of pS421-HTT
- Screened 370 full length kinases for S421 phosphorylation.
- Identified PRKACA as capable of increasing endogenous pS421-HTT levels in human cells, dependent on kinase activity and selective for S421.
Detection in nonhuman primate CSF
- Used SMC assay to detect pS421-HTT in CSF samples from 5 cynomolgus macaques.
- Dephosphorylation abolished pS421 signals, total HTT unaffected, confirming assay specificity.
- Provides proof-of-concept for future clinical biomarker application.
Discussion
- Sensitive SMC assay associated pS421-HTT with phenotypic and CAG length progression in HD mouse model.
- PRKACA identified as a modulator; suggests restoring cAMP levels could affect pS421-HTT phosphorylation.
- Core CSF disease biomarkers (including phospho-tau in AD) show biomarker quantification relevance.
- CSF is a promising yet challenging biomarker matrix in HD due to protein variability and low abundance.
- This work enables potential monitoring of pS421-HTT status in human CSF for disease association studies.
The development of an ultrasensitive immunoassay for the detection of huntingtin phosphorylation at S421 represents a significant advancement in the study of Huntington’s disease biology. This innovative approach enables precise longitudinal tracking in preclinical models and establishes the feasibility of quantifying pS421-HTT in cerebrospinal fluid from non-human primates. The findings, now available as a bioRxiv preprint, open new avenues for mechanistic exploration and the establishment of future biomarker strategies for HD. This work underscores the value of collaborative research between IRBM and CHDI Foundation, contributing meaningfully to the pursuit of improved disease understanding and therapeutic solutions.