Mitochondrial modifications indicative of metabolic impairment appear to represent one of the earliest events in neurodegenerative conditions and precede pathological changes or functional impairment. “Indeed several different groups have reported that in mouse models of Alzheimer’s disease, mitochondrial dysfunction is observable before pronounced pathology appears, indicating early causative mitochondrial defects,” says Gail V.W. Johnson, Ph.D., professor of anesthesiology and of pharmacology and physiology in the mitochondrial research and innovation group at the University of Rochester Medical Center.
The Johnson lab focuses on understanding early cellular modifications in neurodegenerative conditions. In Huntington’s disease, CAG expansions in a ubiquitously expressed protein, huntingtin, cause neuronal loss in the striatum, but the molecular mechanism(s) responsible for this selectivity and the early events causing cellular damage are poorly understood. Research in the Johnson lab supports the hypothesis that mitochondrial dysfunction is a contributing factor to neuronal pathology.
Striatal cells derived from a Huntington’s disease mouse model showed increased sensitivity to mitochondrial complex II inhibition compared to striatal cells from wild type mice, and they also exhibited mitochondrial calcium-handling defects and impaired respiration. Other groups have shown that mitochondria from Huntington’s disease patients and mouse models undergo permeability transition pore opening at significantly lower calcium levels than control mitochondria and, overall, increasing evidence implicates mitochondrial abnormalities as key in the pathogenic process.
In Alzheimer’s disease, accumulation of amyloid beta, the primary component of neuritic plaques that represent the neuropathological hallmark of this condition, was recently shown to increase mitochondrial fragmentation, and there is evidence that tau proteins, which form the neurofibrillary tangles observed in the brain during the course of the disease, mediate amyloid beta toxicity.
Furthermore, a recent report established a link between increased cytosolic calcium levels and mitochondrial fission. “Given that dysregulation of calcium has been associated with Alzheimer’s and Huntington’s diseases, this could be a common contributing factor to the mitochondrial dysfunction that occurs in these diseases; however, further studies are needed,” explains Dr. Johnson.
The combined and integrated use of multiple experimental approaches provides an important driving force that fuels scientific advances. “Genomics and pharmacogenetics are here today. The largest barrier will be the understanding of which genetic variations are important and how they affect protein expression or function. The average person utilizes about 40 kg of ATP in a day. Understanding allelic differences in any of the 18 genes encoding subunits of the ATP synthase will be challenging, but important,” says David M. Mueller, Ph.D., professor of biochemistry and molecular biology at the Chicago Medical School of Rosalind Franklin University.
To gain structural and functional insight into the mitochondrial ATP synthase and the coupling mechanism that converts proton translocation energy into conformational changes resulting in ATP synthesis, the Mueller lab uses a combination of genetics, biochemistry, and structural biology. Two approaches, one that introduces specific mutations known to affect coupling and examines their effect on enzyme biochemistry and structure, and its reverse, which makes structure-based predictions and subsequently examines the impact of individual mutations on coupling, both emerged as important instruments in dissecting mitochondrial biology.
“At this time, crystallizing the entire ATP synthase is an important goal, and we are making progress on that,” says Dr. Mueller. Previously, the Mueller group provided a high-resolution crystal of the yeast mitochondrial F1-ATP synthase which, in combination with the only other previously solved structure, that of the bovine enzyme, explained mechanistic details involved in the conformational changes during ATP synthesis.
“Understanding mitochondrial biology in higher organisms is important,” says Michael Ryan, Ph.D., associate professor of biochemistry at La Trobe University in Melbourne. “Most of the early work has used yeast, which is a great model organism, to look at basic processes. However, in humans, mitochondria appear to be involved in so many more processes that can affect neighboring cells. This includes apoptosis and damage by a generation of reactive oxygen species.”
At Gordon Research Conference’s “Molecular and Cellular Bioenergetics” meeting to be held in June, Dr. Ryan will present recent advances his group made in characterizing new complex I assembly factors. Human complex I, or NADH: ubiquinone oxidoreductase, located in the mitochondrial outer membrane, comprises 45 different subunits, of which only seven are encoded by the mitochondrial DNA. The remaining 38 are encoded by the nuclear genome and imported into the mitochondria for assembly.
The lack of a crystal structure for the ~1 MDa complex I, along with its absence in yeast, explain why relatively little is known about its biology. However, isolated complex I defects were estimated to represent the most common group of mitochondrial diseases, and many are lethal shortly after birth or in early childhood. The Ryan group characterized the intermediates and assembly pathways as different complex I subunits, and in collaboration with David Thorburn, Ph.D., associate professor at the Murdoch Children’s Research Institute in Melbourne, examined almost 100 different cell lines originating from patients with isolated complex I deficiency, half of which did not harbor mutations in any complex I subunit-encoding genes.
Screening these cell lines with antibodies against CIA30 (complex I intermediate associated protein), an assembly factor initially identified in the fungus Neurospora crassa, previously led to the identification of the first patient with CIA30 deficiency, exhibiting an ~70% decrease in complex I levels that were restored to near normal after wild type CIA30 was expressed in the patient’s cultured cells. “I think we are going to find a lot of new proteins involved in complex I assembly,” predicts Dr. Ryan. “Defects in complex I are thought to generate damaging ROS and cause cell death. This area needs to be looked at in closer detail.”