The Role of Dynamin Like Protein 1 in Parkinson's Disease

Thesis (Ph.D.)--University of Washington, 2013Parkinson's disease (PD) is a neurodegenerative disease diagnosed by the presence of various motor symptoms, which result from loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In addition to these motor defects, numerous non-motor symptoms occur and appear prior to the onset of clinical symptoms. Significant information implicates mitochondrial dysfunction in the pathogenesis of PD, with recent evidence showing changes in mitochondrial dynamics may be involved. Although the role of astrocytes has become increasingly recognized as an important factor in promoting neuronal health, their contributions towards PD have yet to be fully realized. This thesis focuses on various aspects related to detection of PD in patients as well as mechanisms of PD that occur through mitochondrial defects, which could represent targets of therapy. Chapter 2 considers the current state of biomarkers in PD and how the development of preclinical markers could be achieved to potentially allow for therapies aimed at preventing neuronal loss. Chapter 3 focuses on mitochondrial involvement in PD and how therapies may act to alleviate mitochondrial deficits. Chapters 4 and 5 examine how astrocytes and excitotoxicity play a role in PD particularly, due to changes in mitochondrial dynamics related to the fission promoting protein dynamin like protein 1 (Dlp1), and how the downstream effects of this could be a target of therapy. Using human tissue and following previous mass spectrometry data, Dlp1 expression was demonstrated to be decreased in the SNpc of PD patients. This decrease occurred in both neurons and astrocytes within the SNpc, a finding that was extended to the same cell types in the frontal cortex of patients without observable cortical degeneration. In pursuing the effects of this decrease in astrocytes, it was observed that knockdown of Dlp1 resulted in extensive interconnection and elongation of mitochondria, combined withan impairment in their movement and localization. Further, knockdown of Dlp1 in astrocytes hindered their ability to protect against the excitotoxic effects of glutamate, which was protected against by blocking NMDA receptors. No changes in expression or localization of the major astrocytic glutamate transporters were observed. Instead, these effects can be tied back to differences in intracellular Ca2+ that occur in response to glutamate, as the intracellular Ca2+ levels were elevated in astrocytes after Dlp1 was knocked down. This was due to impaired mitochondrial buffering of Ca2+ that originates from the extracellular space. These results identify a novel mechanism of mitochondrial dysfunction due to alterations in dynamics, in astrocytes, as a means through which neurodegeneration in PD could develop. Further, the results that Dlp1 is decreased in the cortex prior to the appearance of degeneration indicates that depression of Dlp1 expression is an early event in PD. They also show that targeting excitoxicity could be an effective means of alleviating PD. Such therapies may prove to be effective in preventing neuron loss, if treatment is administered prior to the onset of clinical symptoms, which is dependent upon the development of preclinical biomarkers

Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis

Abnormal oxidative metabolism in a quiet genomic background underlies clear cell papillary renal cell carcinoma

While genomic sequencing routinely identifies oncogenic alterations for the majority of cancers, many tumors harbor no discernable driver lesion. Here, we describe the exceptional molecular phenotype of a genomically quiet kidney tumor, clear cell papillary renal cell carcinoma (CCPAP). In spite of a largely wild-type nuclear genome, CCPAP tumors exhibit severe depletion of mitochondrial DNA (mtDNA) and RNA and high levels of oxidative stress, reflecting a shift away from respiratory metabolism. Moreover, CCPAP tumors exhibit a distinct metabolic phenotype uniquely characterized by accumulation of the sugar alcohol sorbitol. Immunohistochemical staining of primary CCPAP tumor specimens recapitulates both the depletion of mtDNA-encoded proteins and a lipid-depleted metabolic phenotype, suggesting that the cytoplasmic clarity in CCPAP is primarily related to the presence of glycogen. These results argue for non-genetic profiling as a tool for the study of cancers of unknown driver

The value of validated vulnerability data for conservation planning in rapidly changing landscapes

Data needed for informed conservation prioritization are generally greater than the data available, and surrogates are often used. Although the need to anticipate threats is recognized, the effectiveness of surrogates for predicting habitat loss (or vulnerability) to land-use change is seldom tested. Here, we compared properties of two different vulnerability surrogates to validated vulnerability—validated prediction of habitat conversion based on a recent assessment of land-use change. We found that neither surrogate was a particularly effective predictor of vulnerability. Importantly, both surrogates performed poorly in places most imminently threatened with habitat conversion. We also show that the majority of areas protected over the last two decades have low vulnerability to the most active threatening process in this biome (habitat conversion). The contrary patterns of vulnerability and protection suggest that use of validated vulnerability would help to clarify protection needs, which might lead to the improvement of conservation decisions. Our study suggests the integration of validated vulnerability into conservation planning tools may be an important requirement for effective conservation planning in rapidly changing landscapes. We apply our results to discuss the practical considerations and potential value of incorporating validated vulnerability into conservation planning tools both generally and in the context of New Zealand’s indigenous grasslands