Microdevices for Isolating Single Axons for the Study of Axonal Transport in Dopaminergic Neurons
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Axonal degeneration plays an important role in the etiology of major central nervous system: CNS) neurodegenerative disorders such as Parkinson's disease: PD) but investigations into the mechanisms underlying this important process has been limited. The goal of this dissertation was to design and demonstrate the use of a compartmented microdevice which allows for the isolation and study of axonal processes such as mitochondrial transport away from cell neuronal bodies. To achieve this goal, a PDMS microdevice comprised of two large chambers connected by a parallel array of microchannels successfully achieved separation of axons from the soma and allows tracking of transport along oriented axons. To create an in vitro model of PD, dopaminergic axons from midbrain cultures were isolated within the microchannels away from the cell bodies. To determine the best method for imaging intracellular organelles, cells were transduced with a mitochondria targeted DsRed2 lentiviral vector or with a mitochondria specific dye, Mitotracker Deep Red. While both methods successfully labeled the mitochondria, the dye, created significant fluorescent background noise which made measuring mitochondria movement difficult. Using the microdevice, the effects of 6-hydroxydopamine: 6-OHDA), one of the most commonly used toxins for modeling PD, on axonal transport was then investigated on GFP labeled midbrain neurons cultured within the microdevice. 6-OHDA quickly induced mitochondrial transport dysfunction in both GFP and non-GFP labeled axons. Transport of synaptic vesicles were also blocked by the effects of 6-OHDA. 6-OHDA also induced the fragmentation of microtubules and the induction of autophagy but these processes came at a much later time point compared to transport inhibition. These toxin effects on mitochondrial transport were blocked by the addition of SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride: MnTBAP), as well as the anti-oxidant N-acetyl-cysteine: NAC). However, addition of calcium ion chelator, ethylene glycol tetraacetic acid: EGTA), did not restore mitochondrial transport. This study suggests that transport dysfunction occur early and may play a significant role in inducing axonal degeneration in response to 6-OHDA treatment. Overall, the dissertation shows that the microdevice is an excellent tool for studying axonal degenerative processes in the CNS.
Lu, Xi, "Microdevices for Isolating Single Axons for the Study of Axonal Transport in Dopaminergic Neurons" (2013). All Theses and Dissertations (ETDs). 1146.
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Permanent URL: http://dx.doi.org/10.7936/K7FT8J1N