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Updated: May 10, 2025
Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System
Published on: December 10, 2021
Kinetin mediated mutant huntingtin phosphorylation restores multiple dysregulated pathways in a cell line model of
Rajubhai Dabhi1, Ragi Mehta1, Dhruvi Kakadiya1
1Neurobiology and Molecular Diagnostics Lab., Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Sayajigunj, Vadodara, Gujarat 390002, India.
Insights
Kinetin treatment promotes huntingtin (HTT) N-terminal phosphorylation, preventing mutant HTT aggregation. This phosphorylation rescues cellular functions, reduces stress, and improves cell survival, highlighting a promising therapeutic target for Huntington
Area of Science:
- Neurodegenerative disease research
- Molecular biology of protein aggregation
- Cellular pathway dysregulation in disease
Background:
- Huntington's disease (HD) stems from CAG repeat expansion in the huntingtin gene (HTT), leading to mutant HTT (mHTT) aggregation and neurodegeneration.
- N-terminal huntingtin (N-HTT) phosphorylation regulates HTT localization, structure, aggregation, clearance, and toxicity.
- Previous research focused on Ser13/Ser16 phosphorylation's effect on aggregation, with downstream pathway impacts largely unexplored.
Purpose of the Study:
- To investigate the downstream effects of kinetin-induced N-HTT phosphorylation on cellular pathways in a Huntington's disease model.
- To evaluate the therapeutic potential of targeting N-HTT phosphorylation for Huntington's disease.
Main Methods:
- Utilized an inducible cell line model expressing mutant HTT exon 1 with 150 polyglutamine repeats (HD150Q).
- Administered kinetin to induce phosphorylation at Ser13 and Ser16 of N-HTT.
- Assessed protein aggregation, ATP levels, gene transcription, mitochondrial reactive oxygen species (ROS), ER stress markers, and cell survival.
Main Results:
- Kinetin induced N-HTT phosphorylation, preventing mHTT aggregation and resolving pre-formed aggregates.
- Kinetin treatment rescued ATP levels, normalized gene transcription, reduced mitochondrial ROS, and restored mitochondrial function.
- Significantly reduced ER stress markers at multiple levels, leading to improved cell survival.
Conclusions:
- N-terminal HTT phosphorylation is a key regulator of mHTT aggregation and downstream cellular pathology.
- Kinetin-induced phosphorylation mitigates key Huntington's disease cellular defects, including mitochondrial dysfunction and ER stress.
- Targeting N-HTT phosphorylation represents a promising therapeutic strategy for Huntington's disease.
Abstract:
Huntington's disease (HD) is a fatal neurodegenerative disease caused by CAG trinucleotide repeat expansion in the huntingtin gene (Htt) resulting in an expanded polyglutamine (polyQ) tract in the huntingtin (HTT) protein. The expanded polyQ alters structure of HTT making it susceptible to aggregation. The expression of mutant HTT (mHTT) causes dysregulation of several key cellular pathways in neuronal cells resulting in neurodegeneration. Recent studies have demonstrated phosphorylation of the N-terminal domain of the huntingtin (N-HTT) protein as an important regulator of its localization, structure, aggregation, clearance and toxicity. Most studies have focused on the effect of phosphorylation of Ser13 and Ser16 in N-HTT on protein aggregation and reported a drastic reduction in aggregation. However, the downstream impact of this phosphorylation status on key cellular pathways is largely unexplored. Utilizing an inducible cell line model for expression of Exon 1 fragment of mHTT bearing 150 polyglutamine repeats (HD150Q), we demonstrate that kinetin induced phosphorylation at Ser13 and Ser16 of N-HTT resulted in prevention of aggregation as well as resolution of preformed aggregates. Furthermore, kinetin treatment led to rescue of ATP levels and transcription of key genes as well as significant reduction in mitochondrial ROS levels restoring mitochondrial function. Notably, ER stress markers were significantly reduced at transcriptional, translational and post-translational levels. Restoration of mitochondrial function and mitigation of ER stress lead to significant improvement in cell survival. These findings further strengthen the view that HTT N-terminal phosphorylation is a promising therapeutic target for HD.
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