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The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons.

Lu X, Kim-Han JS, Harmon S, Sakiyama-Elbert SE, O'Malley KL - Mol Neurodegener (2014)

Bottom Line: This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles.The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process.Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Washington University in Saint Louis, 1 Brookings Drive, Campus Box 1097, St, Louis, MO 63130, USA. sakiyama@wustl.edu.

ABSTRACT
6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment.

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6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p < 0.05 versus control.
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Figure 4: 6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p < 0.05 versus control.

Mentions: Mitochondria are not the only cargo being transported along the axon. Using standard bright-field microscopy, it is common to see many particles moving bi-directionally along the axon. However, when assessing particle movement in our microchannels, the particles tend to blend into the shadow of the microchannels, as axons adhere to the channel sides, hence particle movement cannot be measured using a standard bright-field microscopy. Therefore, to determine whether 6-OHDA specifically disrupts mitochondrial transport or whether it may affect transport of other axonal cargo, movement of synaptic vesicles was assessed with a synaptophysin-cerulean marker. Previous reports from this lab showed that synaptophysin-cerulean marked small rapidly moving vesicles that did not co-localize with mitochondria [10]. Similar to the decrease in mitochondrial motility, after 30 minutes of treatment with 6-OHDA the movement of synaptic vesicles in both the anterograde and retrograde direction was reduced by 60-70% (Figure 4). Due to the low number of moving particles, meaningful velocity data could not be obtained from measuring the remaining motile particles. These findings show that 6-OHDA affects axon transport machinery resulting in decreased axonal transport of two important cargoes, synaptic vesicles and mitochondria.


The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons.

Lu X, Kim-Han JS, Harmon S, Sakiyama-Elbert SE, O'Malley KL - Mol Neurodegener (2014)

6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p < 0.05 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016665&req=5

Figure 4: 6-OHDA also decreases synaptic vesicle movement in DA axons. A) DA-GFP cultures (Top panels) in microdevices were transduced with Syn-Cer lentivirus (Middle panels) at DIV2. Vesicular movement was assessed on DIV12–13 before and after toxin treatment. Resulting kymographs are shown below. Because of the smaller size of vesicular particles and the relative “dimness” of the cerulean emission, tracks of moving particles are shown in bottom panels for clarity. Red represents retrograde movement whereas blue is anterograde trafficking. B) Quantification of moving vesicles was determined as described in Materials and Methods. Scale bar: 5 μm. Mean ± SEM, total of 8 (control) and 8 (6-OHDA-treated) axons from 5–7 devices per group. * indicates p < 0.05 versus control.
Mentions: Mitochondria are not the only cargo being transported along the axon. Using standard bright-field microscopy, it is common to see many particles moving bi-directionally along the axon. However, when assessing particle movement in our microchannels, the particles tend to blend into the shadow of the microchannels, as axons adhere to the channel sides, hence particle movement cannot be measured using a standard bright-field microscopy. Therefore, to determine whether 6-OHDA specifically disrupts mitochondrial transport or whether it may affect transport of other axonal cargo, movement of synaptic vesicles was assessed with a synaptophysin-cerulean marker. Previous reports from this lab showed that synaptophysin-cerulean marked small rapidly moving vesicles that did not co-localize with mitochondria [10]. Similar to the decrease in mitochondrial motility, after 30 minutes of treatment with 6-OHDA the movement of synaptic vesicles in both the anterograde and retrograde direction was reduced by 60-70% (Figure 4). Due to the low number of moving particles, meaningful velocity data could not be obtained from measuring the remaining motile particles. These findings show that 6-OHDA affects axon transport machinery resulting in decreased axonal transport of two important cargoes, synaptic vesicles and mitochondria.

Bottom Line: This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles.The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process.Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Washington University in Saint Louis, 1 Brookings Drive, Campus Box 1097, St, Louis, MO 63130, USA. sakiyama@wustl.edu.

ABSTRACT
6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment.

Show MeSH
Related in: MedlinePlus