Optineurin-mediated Autophagy in Environmental Models of Parkinson’s Disease

John Pierce Wise, Purdue University

Abstract

Parkinson’s disease is a complex, progressive neurodegenerative disease that has a poorly understood etiology and no known cure. Epidemiological studies suggest the vast majority of cases are likely due to interactions between genetic susceptibilities and exposure to external factors (e.g. environmental chemicals) synergistically contributing to disease development. Pesticides are the strongest environmental link and have been utilized in vivo and in vitro to robustly replicate behavior, pathology, and cellular and molecular impairments observed in patients. Decades of research has revealed a variety of impaired cellular and molecular mechanisms, though it is still unclear which are the most critical or in what order they occur. Intriguingly, the protein optineurin, which is genetically linked to amyotrophic lateral sclerosis and glaucoma, is known to be involved in many of these impaired mechanisms. Moreover, a recent genome-wide association study found the M98K mutation is a risk factor for development of Parkinson’s disease. Increasing evidence suggests a link between Parkinson’s disease and glaucoma, with Parkinson’s patients exhibiting an increased likelihood of developing glaucoma, thinned retinal nerve fiber layers, and a variety of other visual impairments. Hence, we proposed that optineurin participates in Parkinson’s disease pathology and may exhibit polymorphisms that increase susceptibility of developing the disease. To investigate this, we used a rat rotenone model of Parkinson’s disease and evaluated changes in optineurin activity in various brain regions implicated in Parkinson’s disease pathogenesis and considered the contribution of optineurin mutation or depletion to Parkinson’s-like brain pathology. We initially determined that optineurin is expressed in the dopaminergic neurons of the substantia nigra pars compacta, and its expression here appears to be more robust than many other regions by qualitative analysis. Then we considered how optineurin expression changes after rotenone exposure. Across all time points considered (24 h, 5 d, or end-stage parkinsonian phenotype), optineurin expression was significantly elevated. We also found optineurin colocalized with LC3, a critical autophagy-related protein, and the number of colocalized puncta was significantly increased after 5 d rotenone exposure. Such an accumulation of mature autophagosomes has been repeatedly observed in Parkinson’s disease models, and is believed to derive from impaired lysosomal fusion. Finally, in our initial investigation we demonstrated a shift in alpha-synuclein expression from a dispersed, cytosolic expression to a more punctate expression and these puncta were colocalized with optineurin. We believe optineurin colocalizes with alpha-synuclein to target aggregates for degradation by autophagy, but this will need to be verified by further investigation. In the third chapter, we proposed autophagic dysfunction occurs ahead of alpha-synuclein aggregation, and expanded our investigation of optineurin and autophagic dysfunction into four other brainstem regions implicated in preclinical Parkinson’s disease: the dorsal motor vagal nucleus, raphe, locus coeruleus, and pontine tegmental nucleus. Again, we measured optineurin and LC3 expression, mean number of puncta per cell, and colocalized puncta. Furthermore, we considered the percent of optineurin and LC3 puncta that were colocalized (i.e. of the total number of optineurin or LC3 puncta, what percent are colocalized). Collectively, these data suggested impaired lysosomal fusion across multiple regions and multiple time points, as well as impaired binding between optineurin and LC3. We found significantly decreased percent of colocalized puncta for both LC3 and optineurin in most regions and time points considered after rotenone exposure when compared to control. Finally, when we considered the mean number of LC3 puncta per cell in control animals across all regions analyzed, our data show significantly fewer puncta in dopaminergic neurons of the substantia nigra pars compacta and locus coeruleus. These data suggest autophagic capacity may be another limiting factor for dopaminergic neurons ability to cope with cellular stress. Chapter 4 investigates the potential role of optineurin in Golgi fragmentation, which has been observed as an early event in multiple neurodegenerative diseases. Our data showed presence of Golgi fragmentation after rotenone exposure with optineurin colocalizing to fragments, but quantitative analyses were inconclusive regarding the amount of Golgi fragmentation present. Chapter 5 begins to consider the potential for the E50K optineurin mutation or optineurin knock-out contributing to development of Parkinson’s disease. To address this, we considered the signal intensity of tyrosine hydroxylase in the striatum of optineurin transgenic or wild type mice. These preliminary results show decreased intensity of striatal tyrosine hydroxylase in optineurin transgenic mice when compared to control, suggesting optineurin expression is important for nigral dopaminergic neuron survival. In sum, our data is the first to present optineurin as a potentially important protein in the pathogenesis of Parkinson’s disease. Further investigations of optineurin’s role in gene-environment interactions of Parkinson’s disease are warranted and will likely reveal novel mechanisms of pathogenesis and disease progression.

Degree

Ph.D.

Advisors

Cannon, Purdue University.

Subject Area

Toxicology

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS