30 September 2008. The tiny fruit fly Drosophila has served researchers well to unravel the function of genes linked to neurodegeneration in Parkinson disease. Two years ago, several groups used the model to link the fly equivalents of two Parkinson genes, pink1 (PTEN-induced putative kinase 1) and parkin (a ubiquitin protein ligase) into a common biochemical pathway that helps to maintain healthy mitochondria (Park et al., 2006; Clark et al., 2006; Yang et al., 2006). Now, Alexander Whitworth, of the University of Sheffield in the United Kingdom, along with Jeffrey Lee and Angus McQuibban at the University of Toronto in Canada, have used a fly model of neurodegeneration in the eye to enlarge the roster of genes in that pathway, adding two new proteases. Their results suggest that a mitochondrial serine protease, Omi, sits downstream of Pink1, but in a parallel pathway to Parkin. In addition, they find that the intramembrane protease Rhomboid-7 acts upstream to cleave both Pink1 and Omi on the inner mitochondrial membrane. The paper appears in the September issue of Disease Models and Mechanisms, a new monthly published by the Company of Biologists. The journal will emphasize studies in non-traditional models of human disease, such as flies, yeast, and worms.
In other Parkinson-related news, two papers in this week’s PNAS online address the vulnerabilities of dopaminergic neurons that are lost in the disease. One, from Zhengui Xia and colleagues at the University of Washington, Seattle, contradicts the dogma that inhibition of mitochondrial respiratory chain complex I is a key step in the selective loss of dopaminergic neurons in response the toxins rotenone, MPP+, or paraquat. The second paper, a new mouse model of tau pathology, developed by Jurgen Gotz and colleagues at the University of Sydney in Australia, suggests that cell dysfunction and Parkinsonian symptoms can result from alterations in axonal transport.
First, the fly work. Whitworth and colleagues begin by showing that overexpression of Pink1 in Drosophila eye causes a disorganization of the compound lens units leading to a rough eye phenotype. Parkin was required for the Pink1 effects, serving as a downstream effector, in agreement with earlier genetic studies in flies.
To flesh out this skeletal scheme, the researchers used the eye assay to test two more genes for interactions with pink1 and parkin. The first, the high temperature requirement A2 gene (HTRA2, also called OMI), encodes a serine protease and has been found mutated in patients with sporadic PD (Strauss et al., 2005). The new work revealed that overexpressed Drosophila omi interacts with pink1 to cause a more severe eye phenotype. Like parkin, omi acted downstream of pink1. Surprisingly, the parkin and omi genes did not affect each other, suggesting that they comprise parallel pathways emanating from Pink1. Consistent with this idea, removal of either gene partially reversed the rough eye phenotype due to pink1 overexpression, while removal of both was required to completely reverse the pink1 effects.
The second gene, rhomboid-7, attracted the researchers’ interest because mutants have a similar phenotype to pink1 and parkin mutants. Rhoboid-7 is the homolog of the human mitochondrial protease Parl (presenilin-associated rhomboid-like protease). Whitworth and colleagues found that overexpression of rhomboid-7 induced the rough eye phenotype, which was suppressed by the removal of pink1, parkin, or omi. Those results placed rhomboid-7 squarely upstream of all three proteins.
“Where before just two genes linked to the pathway, we’ve now identified two more genes and been able to determine where they lie in that pathway. We are getting a much better idea of what this pathway looks like and the complexity of its structure,” Whitworth told ARF.
Biochemical work gave some clues to how the expanded pathway might work. Rhomboid-7 and its homolog Parl are both mitochondrial intramembrane proteases, functionally similar to the presenilins. Both Pink1 and Omi are inner mitochondrial membrane proteins that are processed to smaller forms in vivo. The investigators show that rhomboid-7 is associated with both proteins in cells, and is necessary to process both to smaller forms. The cleavage reactions seem to be important for the activity of the pathway, as a catalytically inactive from of Rhomboid-7 has a much-reduced effect in the rough eye model.
While the involvement of regulated intramembrane proteolysis in a PD pathway is reminiscent of the role of presenilin in production of amyloid-β in Alzheimer disease, it is too early to tell if there is a true parallel there. “The two are not directly comparable, but the similarities are certainly interesting,” Whitworth said. “It’s unclear at the moment whether the function of rhomboid cleaving pink1, for example, causes anything analogous to [the production of Aβ], but certainly the general similarity of this type of biological mechanism for regulating these membrane-bound proteins is striking. One advantage it gives us is that we can take the knowledge from γ-secretase and Aβ production and turn that to pink1 and omi and ask how Rhomboid-7/Parl regulates their function.” The involvement of PARL in PD also remains to be seen. Whitworth reports that his group is currently searching for PARL mutations in PD patients.
The Pink1/Parkin pathway is linked to mitochondrial biogenesis, and its importance supports the long-standing idea that PD is a mitochondrial disease. In particular, it is widely surmised that inhibition of mitochondrial respiratory complex I is a critical event in the death of dopaminergic neurons in PD. However, new data from Zhengui Xia and colleagues at the University of Washington in Seattle challenges that notion. The study, published in PNAS online on September 23, suggests that lack of complex I activity does not cause dopaminergic cell death, nor does inhibition of complex I explain the sensitivity of the cells to the mitochondrial toxins/complex I inhibitors rotenone and MPP or the suspected inhibitor paraquat.
To look at the role of complex I in dopaminergic neuron death, first author Won-Seok Choi and coworkers made mice with a mutation in Ndufs4, a gene that encodes one of the 46 subunits of complex I and a protein required for complex assembly and activity. Similar mutations in humans are lethal in infancy, and unsurprisingly, the mice die soon after birth. In cultures of primary mesencephalic cells, there was no detectable complex I activity. Nonetheless, the cultures contained normal amounts of tyrosine hydroxylase-positive (TH+) dopaminergic neurons. The cells also looked morphologically and biochemically normal, with no excess apoptosis or generation of reactive oxygen species. The results indicate that loss of complex I activity on its own is not sufficient to induce dopaminergic cell death.
Surprisingly, lack of complex I did not affect the cells’ sensitivity to inhibitors. The knockout cells were not protected from rotenone, the 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine metabolite MPP+, or paraquat, all of which selectively killed dopaminergic neurons in the cultures. In fact, rotenone appeared to be more toxic in the knockout cells. While the researchers cannot rule out that there is still some partial form of complex I targeted by inhibitors, additional data points to a minor role for such a complex in energy production in knockout cells. (For more on this topic, see comment below by David Simon).
The work “suggests that there is complex I-independent, intrinsic property of dopaminergic neurons that makes them mores susceptible [to the three chemicals],” the authors write. But what could that be? They speculate that for rotenone, alternative targets could include microtubule destabilization, vesicle accumulation, and/or oxidative damage.
The idea that other functions go awry and cause degeneration of dopaminergic neurons is supported by a paper from Jurgen Gotz and colleagues at the University of Sydney in Australia. First authors Lars Ittner, Thomas Fath, and Yazi Ke describe a mouse model expressing a mutated human tau protein (K369I) that in humans causes frontotemporal dementia. The protein is expressed in the substantia nigra, and young mice, which do not yet have neuron loss, show Parkinson-like motor symptoms that were reversed by L-dopa treatment. Later, the mice develop age-dependent loss of dopaminergic neurons.
The researchers found that the early dysfunction of dopaminergic neurons resulted from a selective impairment of anterograde axonal transport of specific cargoes, including tyrosine hydroxylase-containing vesicles and mitochondria. Impairment of TH transport should result in less enzyme in striatal synapses and lower dopamine levels. The transport problems were not specific to dopaminergic neurons, but also occurred in tau-expressing sciatic nerves and led to muscle atrophy. The results indicate that interruption of axonal transport by abnormal tau can cause neuronal dysfunction and early Parkinson’s symptoms preceding neuron death, and thus may be a target for treating parkinsonism in tauopathies.—Pat McCaffrey.
Whitworth AJ, Lee JR, Ho VMW, Flick R, Chowdhury R, McQuibban GA. Rhomboid-7 and HtrA2/Omi act in a common pathway with the Parkinson’s disease factors Pink1 and Parkin. Dis. Model. Mech. 2008 Sep; 1(2-3):168-174.
Choi WS, Kruse SE, Palmiter RD, Xia Z. Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat. Proc Natl Acad Sci U S A. 2008 Sep 23. [Epub ahead of print] Abstract
Ittner LM, Fath T, Ke YD, Bi M, van Ersel J, Li KM, Gunning P, Gotz J. Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia. Proc Natl Acad Sci U S A. 2008 Sep. [Epub ahead of print]