Advancing our understanding of neurodegenerative diseases requires model systems that faithfully recapitulate the biology of human neurons. A recent study by Gandy et al. in the International Journal of Molecular Sciences introduces an innovative luminescence-based platform to explore the role of Parkinson’s disease (PD)-associated genes in living cells. By leveraging human induced pluripotent stem cells (iPSCs) and CRISPR-mediated endogenous tagging, researchers at the Early Drug Discovery Unit at The Neuro (Montreal Neurological Institute-Hospital) at McGill University and Health Canada have created a powerful system for investigating protein expression and function in a physiologically relevant setting.
This work is part of an exciting shift in the field—using human-relevant cell models to replace traditional work horse cell lines. When combined with endogenous gene editing methods, this creates research tools that better model the biology being studied, improving the translation of results to human disease. Using these advanced tools, the authors provide an elegant solution for real-time monitoring of PD-related proteins without reliance on antibody-based detection methods.
iPSCs and Endogenous Tagging: A Physiologically Relevant Approach
Studying neurodegenerative disease mechanisms requires human-relevant models, and iPSC-derived neurons provide an unparalleled opportunity to investigate disease-associated proteins in their native context. The objective of this research was to build iPSC models to gain a deeper understanding of understudied Parkinson’s disease-associated genes, including GBA1, LRRK2, SNCA, and PRKN. A significant challenge in studying newly identified Parkinson’s disease-associated proteins is the limited availability of antibodies that are both highly specific and sensitive enough to detect endogenous protein levels. Traditional methods often depend on protein overexpression, which can affect cellular pathways and misrepresent normal biological processes.
To solve this issue, this study employs CRISPR-based endogenous tagging with HiBiT—a small 11amino acid peptide tag. The HiBiT tag allows sensitive, quantitative luminescence detection when paired with its complementation partner LgBiT, which can be added to cell lysates for endpoint analysis or expressed intracellularly for live-cell approaches. By incorporating the HiBiT tag into the endogenous loci of these PD-associated genes, the team minimized the impact on native cellular biology, while creating research tools that can be used to study endogenous protein characteristics with high sensitivity and precision.
The researchers took this a step further by integrating LgBiT into a safe-harbor locus in the master iPSC line under the control of a doxycycline-inducible promoter. Incorporation of intracellular LgBiT allows for real-time, live-cell quantification or bioluminescent imaging of the HiBiT-tagged proteins. By expressing LgBiT with an inducible promoter, the luminescent signal can be turned on only when needed, preserving native protein expression patterns while allowing for precise temporal control over detection. This level of biological relevance makes these models particularly valuable for dissecting disease mechanisms and testing potential therapeutics.
For researchers who prefer a non-integrating approach, Promega’s new ViaScript™ LgBiT mRNA Delivery System offers a flexible and transient alternative method for expressing intracellular LgBiT. Similar to an inducible promoter system, this approach enables researchers to introduce LgBiT and activate live-cell analysis of HiBiT-tagged proteins precisely when needed. The mRNA delivery method ensures consistent and tunable LgBiT expression, providing researchers with an additional tool to analyze HiBiT-tagged protein dynamics in live-cell models.
Applications for Parkinson’s Disease Research
This study is particularly exciting for neurodegenerative disease research, as it focuses on PD—a disorder characterized by the progressive loss of dopaminergic neurons. The team validated their luminescence system by HiBiT-tagging GBA1, a gene linked to both PD and Gaucher disease, and monitoring its activity in iPSC-derived neurons. Importantly, they validated the system using ambroxol, a pharmacological chaperone that enhances GCase enzyme levels and is known to increase GBA1 expression levels. As expected, they observed a significant increase in luminescent signal upon treatment of the GBA1-HiBiT neurons, demonstrating that these models are representing expected biology.
By enabling real-time quantification of PD-associated proteins in human neurons, this platform can help researchers explore disease mechanisms or even identify novel drug candidates with greater physiological relevance than traditional models.
Enabling Open Science: A Commitment to Collaboration
Beyond the scientific innovation, this research also represents a commitment to open science. Many of the iPSC lines generated in this study are available through The Neuro’s Open Biobank, ensuring that the broader scientific community can access and build upon these valuable resources. Researchers can access these lines by selecting the IPSC catalogue from the top-left drop-down menu on the repository website. Open access to well-characterized, biologically relevant cell models accelerates discovery, allowing multiple research teams to test hypotheses, validate findings, and develop new therapeutic strategies.
Conclusion
The work by Gandy et al. showcases the power of integrating cutting-edge molecular tools with iPSC technology to develop biologically relevant models of neurodegeneration. As genetic association studies continue to uncover new genes and proteins linked to the development of PD, researchers often face challenges due to the lack of high-quality, validated antibodies, which limits deeper molecular investigations. By employing endogenous HiBiT tagging and inducible luminescence, this system provides a powerful approach for investigating previously understudied Parkinson’s disease-associated proteins in live human neurons, enabling precise and dynamic characterization of their expression and function.
Learn more about HiBiT-tagging technology: https://www.promega.com/resources/technologies/hibit-protein-tagging-system/
References
Gandy, A., Maussion, G., Al-Habyan, S., et al. (2024). An Inducible Luminescent System to Explore Parkinson’s Disease-Associated Genes. International Journal of Molecular Sciences, 25, 9493. DOI: 10.3390/ijms25179493.
Amy Landreman
