Project Overview

“Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive motor impairment and cognitive decline. Mitochondrial dysfunction is a central feature of HD pathophysiology, contributing significantly to neuronal death. Heritable mutant huntingtin protein disrupts mitochondrial dynamics, impairs oxidative phosphorylation and leads to bioenergetic deficits. A key component of this dysfunction involves dysregulation of mitochondrial calcium (Ca2+) homeostasis. In HD, mutant huntingtin enhances Ca2+ uptake into mitochondria, resulting in Ca2+ overload, promoting mitochondrial permeability transition pore (mPTP) opening, loss of membrane potential and activation of cell death signaling pathways.

Most mitochondrial Ca2+ uptake is tightly regulated by the mitochondrial Ca2+ uniporter complex (mtCU), made up of MCU pore forming subunits, MCU dominant negative beta subunits (MCUB), essential MCU regulator (EMRE) and mitochondrial uptake-1/2/3 (MICU1/2/3) gatekeeping proteins. A series of cryo-electron microscopy structures have revealed the fundamental mechanisms underlying mtCU function; however, how post-translational modifications (PTMs), which modulate protein structure and function, affect mtCU remain a major knowledge gap.

Nitric oxide (NO) is a small gaseous signaling molecule that also plays a key role in the pathogenesis of HD. Elevated NO levels, primarily generated by nitric oxide synthases (NOS), contribute to nitrosative stress, which in turn causes mitochondrial dysfunction and damage. One consequence of nitrosative stress is S-nitrosylation, a typically labile post-translational modification (PTM) that occurs on cysteine (Cys) thiol groups. Our lab previously identified a matrix-facing Cys residue in the MCU (i.e. Cys97) as a target of oxidative and nitrosative modifications, alterations that profoundly affected the structure and function of MCU. Given the high sequence and structural similarity between MCU and its paralog MCUB, and the presence of matrix-oriented Cys residues in MCUB, we hypothesize that one or more cysteines in MCUB may also undergo S-nitrosylation. Given our observations with MCU, we posit that this modification will similarly disrupt the structural integrity and/or interaction with MCU, a key negative regulating feature of mtCU function. To test this hypothesis, we plan to express, purify and isolate the matrix-oriented domain of MCUB containing two Cys residues, apply an in vitro S-nitrosylation protocol, use site-directed mutagenesis to block S-nitrosylation in control experiments and characterize how the PTM changes the secondary, tertiary and quaternary structure of the domain.

The intersection of dysregulated Ca2+ handling and NO-mediated stress exacerbates mitochondrial dysfunction, ultimately accelerating neurodegeneration in HD. By elucidating how these pathways interact, particularly through modifications of key mitochondrial regulatory proteins like MCUB, this research aims to uncover fundamental mechanisms driving HD pathology. Targeting mitochondrial Ca2+ signaling and NO-mediated modifications offers a promising avenue for therapeutic and/or diagnostic development. Importantly, this project focuses on core biological processes at the level of protein translation and structure, which are not influenced by sex or gender, ensuring broad relevance of the findings.”

Principal Investigator

Isabella Bu , University of Western Ontario

Partners and Donors

The Huntington Society of Canada