Mitochondrial Group

Area of Research | Background | Projects | Research Approaches | Publications | Contacts

Mitochondrial and Genetics Pathology Group

Ian Trounce

Group Leader Ian Trounce Research Fellows Peter Crouch James Duce Research Assistant Rachel Blake Graduate Student Kathryn Cimdins (PhD)

Area of Research

Neuronal cell death, in particular how protein aggregation may damage neurons by toxic interactions with core mitochondrial functions, and the interaction of genes and environmental toxins in the aetiology of neurodegenerative diseases.

We focus on the commonest age-related neurodegenerative diseases.  An emergent theme linking these diseases is the presence of toxic aggregates of common cellular proteins: amyloid beta in Alzheimer’s disease, Alpha-synuclein in Parkinson’s disease, cytosolic superoxide dismutase in familial ALS, and huntingtin with expanded polyglutamine tract in Huntington’s disease.

• Parkinson's Disease, including early onset forms (Autosomal recessive juvenile Parkinson's; ARJP), Lewy body dementias, and general synucleinopathies.
• Alzheimer's Disease, including frontotemporal dementias (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP).
• Amyotrophic Lateral Sclerosis, including adult-onset motor neuron disease.
• Huntington's Disease, including spino-cerebellar ataxias and adult onset trinucleotide repeat disorders.

Background

Mitochondrial Toxicity in Neurodegenerative Diseases:

Respiratory chain, OXPHOS, ROS

Mitochondria house a large number of important metabolic pathways, many of which are tissue specific.  Our expertise and interest lies in the core mitochondrial function of oxidative phosphorylation, or OXPHOS, which occurs in all mitochondria.  Most cellular ATP is produced by this system, which consists of the respiratory chain protein complexes I to IV, combined with the H+ATPsynthase or complex V.  OXPHOS is also a constant source of by-product reactive oxygen species (ROS), which are detoxified by mitochondrial and cellular antioxidant defenses including MnSOD (mitochondrial SOD or SOD2), Cu/ZnSOD (cytosolic SOD or SOD1), glutathione and glutathionine peroxidase.  Recent findings suggest that ROS are involved in at least some signaling pathways for programmed cell death.  Another recently described role of the respiratory chain is in some oxygen sensing pathways, possibly also using ROS as messengers, leading to regulation of hypoxic inducible factor alpha (HIFa) which is a transcriptional regulator of key genes involved in acute and chronic hypoxic response.

Scheme showing the topological arrangement of the five OXPHOS enzyme complexes (coloured) together with some putative interactions with cell death activators (Bax, Bak, Bid, Bim) and inhibitors (Bcl-2).  Members or the Permeability transition pore (PT pore) are also shown, the voltage dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and (?allosteric) regulators of the pore, the peripheral benzodiazepam receptor (PBR) and cyclophilin D. 

The main sites of ROS generation are thought to be complexes I and III.  Inhibition of electron transport at any complex can increase ROS output due to “short-circuit” of electron flow with univalent reduction of molecular oxygen.

Cell death induction occurs when key intermembrane mitochondrial proteins, including cytochrome c and/or AIF, are allowed to escape the outer membrane into the cytosol.  Controversy remains as to how this is achieved, some favouring permeabilization following Bax recruitment to the membrane, while others favour PT pore induction with consequent swelling and rupture of the outer membrane.

Mitochondria and cell death

Extrinsic death signals such as induced by TNFa or Fas ligation, and intrinsic toxic insults including disordered cytosolic calcium or ROS homeostasis, converge to activate the mitochondrial pathway of cell death.  This leads to mitochondrial membrane potential collapse and the leakage of key mitochondrial proteins into the cytoplasm, ultimately activating the caspase cascade of executioner proteases.

Direct roles for respiratory chain complexes have not yet been defined in programmed cell death, but this may be the next frontier of mitochondrial research.  Our experimental approaches aim to directly investigate this hypothetical linkage by focusing on effects on OXPHOS arising from mutant proteins associated with neurodegenerative diseases.

Interaction with xenobiotics and OXPHOS to cause specific neuronal loss.

Some xenobiotics have created surprising models of neurodegenerative disease.  Thus, the human ‘model’ of drug-induced Parkinsonism resulting from specific inhibition of respiratory chain complex I due to MPTP.  This is also reproduced in mouse and primate models, and a rat model of Parkinsonism was recently described using another classic complex I inhibitor, the common pesticide rotenone.  In all cases a specific loss of dopaminergic neurons is observed.  Respiratory chain complex II inhibitors produce nigrostriatal neuronal loss recapitulating Huntington’s disease.  In human PD there is a well described decrease in complex I activity in affected brain regions, and in HD there is a reduction of complex II.  It is possible that exposures to both xenobiotic and endogenous compounds toxic to OXPHOS function combine with genetic predispositions to result in specific neurodegeneration.

Therapeautic considerations

Some distinctive features of mitochondria make them attractive targets for drug development, e.g. the negative inner charge of the organelle, allowing drug delivery linked to lipophilic cations.  The characteristics of mitochondrial targeting leader peptides are also well known and can be used to deliver larger molecules, into the mitochondrial matrix.

Projects

Novel mouse models of mtDNA-linked OXPHOS dysfunction: Genetic approach

With the collaboration of Carl Pinkert at the University of Rochester, NY, we are producing novel mouse models of mild respiratory chain deficiency.  Some major hurdles have prevented the production, until now, of mouse models of mitochondrial DNA disease.  First, there is no demonstrable recombination in the mitochondrial genome, so conventional transgenic approaches cannot be used.  Second, until the last few years there were no published female embryonic stem (ES) cell lines shown to be germline competent. 

We obtained such a female ES line from Allan Bradley in 2000 and began a novel strategy for producing transmitochondrial ES cell cybrids.  Our approach is to replace the endogenous mouse mtDNA with mtDNAs from progressively more divergent mouse species, resulting in “xenomitochondrial” mice.  Blastocyct injections, chimerae production and breeding are being done by Professor Pinkert in Rochester.  In 2001, we produced the world’s first viable mouse with a completely replaced mitochondrial genome, proof of principle that our approach works.  Despite poor germline competency of the ES cells, in 2004 we have produced our first female germline offspring with replaced mtDNAs.  We have three further transmitochondrial ES cells line constructs underway, one or more of which we anticipate will result in mild mitochondrial disease expression in mice.

When available, we will crossbreed these mice with various neurodegenerative disease mouse lines.  This will invoke a powerful genetic approach to studies of OXPHOS function in the common neurodegenerative diseases.

Parkinson’s disease, a-SN and complex I inhibition

In addition to the toxicological evidence for complex I impairment in Parkinson’s disease mentioned above, there is also some biochemical and genetic evidence that complex I is impaired in Parkinson’s disease patient tissues.  6-OH-Dopa is also a potent complex I inhibitor, and it is possible that disordered dopamine metabolism consequent to mutant alpha-synuclein, for example, may contribute to cell loss through complex I inhibition.

Alzheimer’s disease,  Ab and complex IV inhibition

We are also investigating putative direct effects of the amyloid-beta peptide on the respiratory chain, especially complex IV (also called cytochrome oxidase or COX).  Previous data from our lab and others have indicated that complex IV is preferentially decreased in affected brain regions, and that COX-deficient neurons accumulate in AD patients.

Mitochondria and Neurodegeneration in Amyotrophic Lateral Sclerosis

Present evidence indicates that amyotrophic lateral sclerosis (ALS also known as Motor Neuron disease) is a multifactorial disease in which the selective loss of motor neurones reflects a complex interplay between genetic factors, oxidative stress and excitotoxicity, leading to cell death.  We have been focusing on putative mitochondrial involvement, and the potential for therapies directed at alleviating mitochondrial dysfunction due to toxic gain of function effects of mutant SOD1.

For in vitro studies, we are using one of the best-characterised and genetically relevant mouse models, the G93A SOD1 mouse.  Although only carried in around 2% of all ALS cases, all SOD1 mutants studied in mouse models tend to form aggregates.  This is significant because dozens of different fALS-linked SOD1 mutants have now been reported.  SOD1 is a small protein, forming homodimers housing the catalytic copper and zinc molecules, and is one of the most abundant proteins in motor neurones.  Preliminary data from spinal cord autopsy material from sporadic ALS patients suggests that biochemically-defined SOD aggregates also occur in the human disease (i.e in the absence of SOD1 mutations).  If this is confirmed, our studies in the G93A SOD1 mouse model may shed light on similar pathogenetic mechanisms, and have direct significance for the sporadic human disease. 

Research Approaches (Methods)

We combine biochemical, molecular biology and cell biological approaches.  We can offer student projects using both in vitro and in vivo experimental settings.  Animal work centers on our unique mtDNA mouse model (see above), the G93A SOD1 mouse and rat (in collaboration with Surinder Cheema), and the Alzheimer’s Tg2756 mouse model.  Cell-based projects include the characterization of extent of mitochondrial penetration of aberrant neurodegenerative disease-associated proteins, and effects of these on OXPHOS and ROS production.  Our approach in this case is to use stable transfection of disease-linked proteins into human and mouse ‘neuronal-like’ cell lines, followed by purification of mitochondria and characterization of target protein topology and effects on OXPHOS/ROS using biochemical methods including enzymology, western blotting, immunoprecipitation and pull-down assays.

Particular strengths include:

• cybrid (cytoplasmic hybrid) cell construction to investigate mtDNA effects on ‘control’ nuclear background in cell cultures
• mitochondrial isolation and purification from cultured cells and animal tissues
• enzymological characterization of OXPHOS complexes and in vitro toxicity of disease-associated proteins
• characterization of reactive oxygen (ROS) production coupled with normal and abnormal OXPHOS complexes
• proteomic studies using purified mitochondria

Mitochondrial OXPHOS and Neurodegeneration: Summary

The hypothetical thrust underlying our mitochondrial studies in the Centre for Neuroscience is that neuronal OXPHOS is susceptible to various genetic, neurochemical, age-related and xenobiotic toxic insults.  Impairment of the respiratory chain complexes can reduce ATP output and increase superoxide production.  These effects can converge to activate the mitochondrial cell death pathway in the common neurodegenerative diseases: PD, AD, ALS and HD. 

The major OXPHOS complexes and some of these pathological interactions are depicted, emphasizing the putative role of increased superoxide output from the respiratory chain.  The cell at top indicates how such disturbances may activate the mitochondrial pathway of programmed cell death.

Financial Support

NH&MRC Project grant 145719 (IT), Program grant 208978, Schering AG Berlin, NSV, Prana Biotechnology Ltd, Bethlehem Griffiths Research Foundation, Motor Neuron Disease Foundation, The University of Melbourne.

Recent Publications

PinkertCA, Smith LC, Trounce IA.  Transgenic Animals; modifying the mitochondrial genome.  In: Pond WG, Bell AW (Eds), Encyclopedia of Animal Science, Marcel Dekker Inc., New York (2004)(In press)

Trounce IA and Pinkert CA.  Cybrids in the Study of Animal Mitochondrial Genetics and Pathology. In: Berdanier, CD (Ed.), Mitochondria in Health and Disease, Marcel Dekker Inc., New York (2004)(In press)

Trounce IA, McKenzie M, Cassar CA, Ingraham CA, Lerner C, Dunn DA, Donegan CL, Takeda WK, Pogozelski WK, Howell RL and Pinkert CA.  Development and initial characterization of xenomitochondrial mice.  J Bioenerg Biomembr (In press) (2004)

McKenzie M, Trounce IA Cassar C and Pinkert CA.  Production of homoplasmic xenomitochondrial mice.  Proc Natl Acad Sci (USA)  101:1685-1690. (2004)

McKenzie M, Chiotis M, Pinkert CA and Trounce IA.  Functional respiratory chain analyses in murid xenomitochondrial cybrids expose coevolutionary constraints of cytochrome b and nuclear subunits of complex III.  Mol Biol Evol  20:1117-1124. (2003)

Trounce I, Feeney SJ and Byrne E.  Pathoetiology of Motor Neuron Disease: New insights from genetics and animal models. J Clin Neurosci 10:293-296. (2003)

Setterfield K, Williams AJ, Donald J, Thorburn DR, Kirby DM, Trounce I and Christodoulou J.  Flow cytometry in the study of mitochondrial respiratory chain disorders.  Mitochondrion (2002)1:437-445.

Pinkert CA and Trounce I.  Production of transmitochondrial mice.  Methods(2002) 26:348-357.

Trounce IA.  Genetic control of cellular oxidative phosphorylation and experimental models of defects.  Human Reprod 15(Suppl.2):18-27. (2000)

Trounce I, Schmiedel J, Hsiu-Chuan Y, Hossini S, Brown MD, Olsen G and Wallace DC.  Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells.  Nucleic Acids Res. 28:2164-2170 (2000)

McKenzie M and Trounce I.  Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects. J Biol Chem 275:31514-31519. (2000)

Brown MD, Trounce IA, Jun AS, Allen JC and Wallace DC.   Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778 or 14484 Leber’s Hereditary Optic Neuropathy mtDNA mutations. J Biol Chem 275: 39831-39836. (2000)

Contact Us

For more information regarding student projects, commercial consultation, or collaborations:

i.trounce@unimelb.edu.au

Laboratory Photos