Light-chain split luciferase assay implicates pathological NOTCH3 thiol reactivity in inherited cerebral small vessel disease

Topic: Brain proteins, microglia and lab models

Stereotyped mutations in NOTCH3 drive CADASIL, the leading inherited cause of stroke and vascular dementia. The vast majority of these mutations result in alterations in the number of cysteines in the gene product. However, non-cysteine-altering pathogenic mutations have also been identified, making it challenging to discriminate pathogenic from benign NOTCH3 sequence variants. Here, we present a method for quantitative assessment of NOTCH3 mutants, the light chain split luciferase (LSL) assay. In LSL, NOTCH3 mutant fragments, cloned between a split luciferase open reading frame, are transfected into cells, producing secreted luciferase activity that is dependent on the normal structure of NOTCH3. Insertion of point mutants that cause CADASIL results in significantly lower activity. Using a panel of 47 sequences, we determined the sensitivity and specificity of LSL for pathogenic NOTCH3 mutation discrimination to be 100% and 93%. LSL was also modestly successful in differentiating pathogenic proteins responsible for Marfan's disease and Stiff Skin Syndrome. Two additional parameters from the LSL analysis (TCEP rescue of activity and secretion index) were also shown to be useful in characterizing NOTCH3 mutants. We show that the spacing and primary sequence of the light chain module is an important component of the LSL assay, as a single light chain cysteine is critical for pathogenic sequence discrimination. Furthermore, we show that the activity of CADASIL mutant reporters is amplified by the application of cysteine-reactive iodoacetamide, suggesting that LSL may be deployed to screen for novel compounds that suppress pathogenic conformations of NOTCH3.

Key findings

• The vast majority of these mutations result in alterations in the number of cysteines in the gene product.
• Insertion of point mutants that cause CADASIL results in significantly lower activity.
• Furthermore, we show that the activity of CADASIL mutant reporters is amplified by the application of cysteine-reactive iodoacetamide, suggesting that LSL may be deployed to screen for novel compounds that suppress pathogenic conformations of NOTCH3.

Why it matters

This laboratory study describes a new assay to distinguish harmful NOTCH3 mutations that cause CADASIL, an inherited small vessel disease. It suggests the assay could be used to screen for compounds that suppress harmful NOTCH3 shapes, but this is early laboratory work and no treatment is proven.

Study details

• Evidence strengthLow

Read the original

• DOI: 10.1016/j.jbc.2025.108224
• PubMed: 39864627
• Original source →
• Free full text: Journal of Biological Chemistry →

Full text: free to read — OpenAlex lists this copy as free to read: the link above (checked 6 Feb 1020).

@article{cartee2025-39864627,
  author  = {Cartee, Naw May Pearl and Lee, Soo Jung and Wu, Emily and Sukpraphrute, Richard and Sukpraphrute, Catherine and Greenbaum, Jolie and Wang, Michael M.},
  title   = {{Light-chain split luciferase assay implicates pathological NOTCH3 thiol reactivity in inherited cerebral small vessel disease}},
  journal = {Journal of Biological Chemistry},
  year    = {2025},
  doi     = {10.1016/j.jbc.2025.108224},
  pmid    = {39864627},
  url     = {https://doi.org/10.1016/j.jbc.2025.108224}
}

Studies this one cites

Referenced by this study and also in the alzhub library, so you can read them here.

• Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia Nature · 1996-10-01 · usefulness score 58/100 · Lewy body dementia

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