Angela Poole
Mentor: Leo PallanckBox: 357730
Phone: 206-616-5996
Email: http://www.gs.washington.edu/academics/gradprogram/poole.htm
Angela Poole is interested in Parkinson's Disease (PD), the second most common neurodegenerative disorder in the United States afflicting approximately 1-2% of individuals above the age of fifty-five. While the vast majority of PD cases are sporadic, the mechanisms underlying the neurodegenerative process are most likely shared by the genetic forms of the disease. Loss-of-function mutations in either parkin or PINK1 are responsible for a large fraction of early-onset recessive forms of Parkinsonism, and recent functional analyses have indicated that the two genes are in a common pathway affecting mitochondrial integrity, including the integrity of dopamine neurons in the central nervous system. The parkin gene encodes an E3 ubiquitin ligase which partially localizes to the mitochondria while PINK1 encodes a kinase which localizes to the mitochondrial intermembrane space. It is thought that the build-up of proteins normally targeted to the ubiquitin proteasome system by Parkin is responsible for the toxicity leading to neurodegeneration in parkin-related PD cases. Mutational analyses of highly conserved Drosophila orthologs of these genes indicate that PINK1 acts upstream of Parkin in a common pathway. While the discovery of the PINK1/Parkin pathway represents a major advance, we still know little about the other genetic components in the pathway, how the genetic components interact, or how their dysfunction results in disease.
Transmission electron microscopy images of flight muscle in parkin mutants reveal enlarged mitochondria suggesting the involvement of parkin in the regulation of mitochondrial morphology. The Pallank lab has conducted experiments to determine if there were interactions between parkin and PINK1 and mitochondrial proteins mediating changes in mitochondrial morphology. Mitochondria constantly undergo two opposing processes: fission and fusion. The dynamic regulation of mitochondrial morphology is critical to mitochondrial function, where a shift to either a fused reticulum or a fragmented state leads to disease. Several GTPases play key roles in the mechanics of mitochondrial dynamics. Drp1, Dynamin-related protein 1, is a cytoplasmic protein that assembles with mitochondria and promotes the fission event, and optic atrophy 1 (OPA1) and the mitofusins (Mfn1 and Mfn2) are believed to coordinate fusion of the inner and outer mitochondrial membrane, respectively. We discovered that mutations in drp1 are lethal in a parkin background and largely lethal in a PINK1 background. Conversely, increased dosage of Drp1 and loss-of-function mutations in the fusion-promoting components, OPA1 and Mfn, significantly rescue several parkin and PINK1 mutant phenotypes: thoracic indentations and flight and climbing ability. These findings suggest that Parkin and PINK1 promote fission either by promoting the fission component, Drp1, or inhibiting the fusion components, Mfn and OPA1. These data were recently published in PNAS (105 (5): 1638-1643.) Because of the striking genetic interactions between Drp1 and the PINK1/Parkin pathway and the proteomics experiment identifying Drp1 as being more abundant in wildtype mitochondrial samples than in PINK1 samples, Angela would like to explore the possibility that Drp1 is a direct substrate of PINK1 and/or Parkin. She plans to address this question by using western blot analysis on mitochondrial samples from flies with a FLAG-tagged version of Drp1 expressed in wildtype and PINK1 and parkin mutants.
Angela will also explore the mechanism by which altered fission influences tissue integrity. As a foundation for this work, she is studying the role of this pathway in the nervous system. She will perform experiments to test several non-mutually exclusive models of how defective fission may impact neuronal integrity. One possibility is that defects in fission may disrupt localization of mitochondria at the synapse. Published data from the Bellen lab shows that mutations in drp1 severely impair the localization of mitochondria to the synapse at the neuromuscular junction, and mutants fail to maintain normal neurotransmission during intense stimulation. Impaired mitochondrial localization in dopaminergic neurons could affect calcium buffering at the synapse or ATP levels available for synaptic transmission. She would like to compare the localization of mitochondria in dopaminergic neurons of wildtype, PINK1, and parkin adult fly brains.
Another way that fission may impact neuronal integrity is by protecting against oxidative stress. It has been shown that knocking down fission in cells leads to an increase in reactive oxygen species production. Brain samples from deceased Parkinson's Disease patients have also been shown to have increased levels of oxidative stress. Angela will compare the rate of reactive oxygen species production in Drosophila primary neuronal cell cultures from wildtype flies and PINK1, parkin, drp1 and OPA1 mutants, as well as in cells overexpressing drp1.
Publications
Poole, AC, Thomas, RE et al., 2008. The PINK1/Parkin pathway regulates mitochondrial morphology. PNAS 105 (5): 1638-1643.