Browsing by Author "Kilpatrick, Kiri"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated α-Synuclein
    (Public Library of Science, 2015) Kilpatrick, Kiri; Zeng, Yimeng; Hancock, Tommy; Segatori, Laura; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
    Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-β-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.
  • Thumbnail Image
    Item
    Quantitative Analysis of α-Synuclein Solubility in Living Cells Using Split GFP Complementation
    (Public Library of Science, 2012) Kothawala, Ahmed; Kilpatrick, Kiri; Novoa, Jose Andres; Segatori, Laura; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
    Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells.
  • Thumbnail Image
    Item
    Reprogramming the proteostasis network to prevent the accumulation of alpha-synuclein aggregates
    (2014-03-14) Kilpatrick, Kiri; Segatori, Laura; Gonzalez, Ramon; Silberg, Jonathan J.; Nagrath, Deepak
    Protein misfolding and aggregation characterizes the development of a number of neurodegenerative diseases, such as Parkinson’s, Alzheimer’s and Huntington’s disease. The hallmark of Parkinson’s disease is the formation of proteinaceous inclusions, which consist primarily of α-synuclein (α-syn), a natively unstructured protein with propensity to misfold and aggregate. Cells have evolved sophisticated systems of protein quality control to prevent accumulation of non-native proteins and maintain protein homeostasis. However, the load of misfolded α-syn typically exceeds the capacity of the quality control system. Aberrant accumulation of misfolded α-syn leads to proteotoxic stress, eventually resulting in neurodegeneration. The objective of this project is to investigate chemical and genetic approaches to modulate the protein quality control system and reduce the accumulation of aberrant α-syn species. Studying α-syn aggregation in cells presents a number of challenges mainly due to the limited availability of tools to quantitatively distinguish between different α-syn conformational species within the cellular environment. To address this need, we engineered an in vitro model system based on neuroglioma cells that accumulate α-syn aggregates and developed a set of analytical tools based on the use of aggregation responsive probes to quantify α-syn aggregation in cells. To test whether modulating the protein quality control system affects the accumulation of α-syn aggregates, we investigated a series of complementary approaches aimed at i) enhancing the innate cellular chaperone machinery, which promotes folding and prevents aggregation, and ii) upregulating the autophagy pathway, which mediates clearance of aggregated proteins. We demonstrated that chemical modulation of Hsp70, a ubiquitously expressed molecular chaperone, affects the accumulation of α-syn aggregates. Particularly, the Hsp70 upregulator carbenoxolone was found to reduce α-syn aggregation and prevent α-syn-induced cytotoxicity via activation of the heat shock response. We also found that activation of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, results in enhanced autophagic clearance of α-syn aggregates. We demonstrated that cell treatment with 2-hydroxypropyl-β-cyclodextrin reduces the accumulation of aggregated α-syn specifically by upregulating TFEB-mediated autophagic clearance. These findings lay the foundation for the development of pharmacological strategies to reduce the accumulation of misfolded and aggregated α-syn for the treatment of Parkinson’s disease.