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Axon degeneration and PGC-1α-mediated protection in a zebrafish model of α-synuclein toxicity.

O'Donnell KC, Lulla A, Stahl MC, Wheat ND, Bronstein JM, Sagasti A - Dis Model Mech (2014)

Bottom Line: With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system.The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection.Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.

ABSTRACT
α-synuclein (aSyn) expression is implicated in neurodegenerative processes, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In animal models of these diseases, axon pathology often precedes cell death, raising the question of whether aSyn has compartment-specific toxic effects that could require early and/or independent therapeutic intervention. The relevance of axonal pathology to degeneration can only be addressed through longitudinal, in vivo monitoring of different neuronal compartments. With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system. We therefore expressed human wild-type aSyn in zebrafish peripheral sensory neurons, which project elaborate superficial axons that can be continuously imaged in vivo. Axonal outgrowth was normal in these neurons but, by 2 days post-fertilization (dpf), many aSyn-expressing axons became dystrophic, with focal varicosities or diffuse beading. Approximately 20% of aSyn-expressing cells died by 3 dpf. Time-lapse imaging revealed that focal axonal swelling, but not overt fragmentation, usually preceded cell death. Co-expressing aSyn with a mitochondrial reporter revealed deficits in mitochondrial transport and morphology even when axons appeared overtly normal. The axon-protective protein Wallerian degeneration slow (WldS) delayed axon degeneration but not cell death caused by aSyn. By contrast, the transcriptional coactivator PGC-1α, which has roles in the regulation of mitochondrial biogenesis and reactive-oxygen-species detoxification, abrogated aSyn toxicity in both the axon and the cell body. The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection. Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds.

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Axonopathy is not followed by ‘dying back’ or Wallerian-like degeneration in aSyn-expressing neurons. (A,B) Time-lapse imaging of neurodegeneration. Cells were imaged every 20 minutes beginning 54 hours post-fertilization (hpf). Axons from at least 11 embryos from each group were transected; representative images from aSyn-expressing animals are shown. Time stamps in images are relative to the start of the imaging period. Axonal varicosities were observed (white arrowheads) several hours before cell death. White arrows point to morphological changes indicative of cell death. Inset represents cell body magnified 2×. Asterisk in A indicates separation of the axon from the cell body. Axonal fragmentation (blue arrowheads) usually did not occur before cell death, and was not stereotyped: it did not occur synchronously along the length of the axon, nor in a retrograde direction (yellow arrows point to distal portions of the axon that are still intact). (C,D) Representative images of wild-type (C) and aSyn-expressing (D) axons undergoing WD after transection with a two-photon laser. Axons were transected with a two-photon laser at 2 dpf, and embryos were imaged every 30 minutes for up to 12 hours. Red arrowhead points to site of transection. After injury, in both wild-type and aSyn-expressing axons, fragmentation was synchronous along the length of the transected axon (blue arrowheads). mpa, minutes post-axotomy. (E) There was no difference in the duration of the lag period between transection and fragmentation (WT: 129.1±10.0 minutes, n=11 axons from 11 animals; aSyn: 112.7±11.7 minutes; n=15 axons from 15 animals, P=0.3173). (F) The time between fragmentation and clearance of all axonal debris was not significantly different between the two groups (WT: 58.2±6.9 minutes; aSyn: 69.1±8.3 minutes; P=0.3213). Scale bars: 50 μm.
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f3-0070571: Axonopathy is not followed by ‘dying back’ or Wallerian-like degeneration in aSyn-expressing neurons. (A,B) Time-lapse imaging of neurodegeneration. Cells were imaged every 20 minutes beginning 54 hours post-fertilization (hpf). Axons from at least 11 embryos from each group were transected; representative images from aSyn-expressing animals are shown. Time stamps in images are relative to the start of the imaging period. Axonal varicosities were observed (white arrowheads) several hours before cell death. White arrows point to morphological changes indicative of cell death. Inset represents cell body magnified 2×. Asterisk in A indicates separation of the axon from the cell body. Axonal fragmentation (blue arrowheads) usually did not occur before cell death, and was not stereotyped: it did not occur synchronously along the length of the axon, nor in a retrograde direction (yellow arrows point to distal portions of the axon that are still intact). (C,D) Representative images of wild-type (C) and aSyn-expressing (D) axons undergoing WD after transection with a two-photon laser. Axons were transected with a two-photon laser at 2 dpf, and embryos were imaged every 30 minutes for up to 12 hours. Red arrowhead points to site of transection. After injury, in both wild-type and aSyn-expressing axons, fragmentation was synchronous along the length of the transected axon (blue arrowheads). mpa, minutes post-axotomy. (E) There was no difference in the duration of the lag period between transection and fragmentation (WT: 129.1±10.0 minutes, n=11 axons from 11 animals; aSyn: 112.7±11.7 minutes; n=15 axons from 15 animals, P=0.3173). (F) The time between fragmentation and clearance of all axonal debris was not significantly different between the two groups (WT: 58.2±6.9 minutes; aSyn: 69.1±8.3 minutes; P=0.3213). Scale bars: 50 μm.

Mentions: It has recently been proposed that the axon degeneration observed in PD represents an early, and potentially independent, process in pathophysiology (O’Malley, 2010; Burke and O’Malley, 2012; Jellinger, 2012). In zebrafish neurons expressing aSyn, the percentage of cells with dystrophic axons between 2 and 3 dpf was higher than the percentage of cells that died during that period. To determine whether severe axonopathy always preceded cell death, we conducted time-lapse imaging at 20-minute intervals between 56 and 68 hpf (Fig. 3A,B). In cells that died during the imaging period, the onset of axonal dystrophy (beading or fragmentation) was compared with morphological changes in the soma that herald cell death. In all cases (n=9), focal or diffuse swellings (axonopathy stage 2-3) were seen in axons several hours before cell death (Fig. 3A,B). Axonal fragmentation, however, did not precede apoptotic changes in the cell body (Fig. 3A,B, arrows). Overt axonal breakdown therefore does not proceed directly to the death of the cell body in this model. However, because axonal dystrophy preceded cell death, it is likely that the axonal compartment is more vulnerable to aSyn toxicity.


Axon degeneration and PGC-1α-mediated protection in a zebrafish model of α-synuclein toxicity.

O'Donnell KC, Lulla A, Stahl MC, Wheat ND, Bronstein JM, Sagasti A - Dis Model Mech (2014)

Axonopathy is not followed by ‘dying back’ or Wallerian-like degeneration in aSyn-expressing neurons. (A,B) Time-lapse imaging of neurodegeneration. Cells were imaged every 20 minutes beginning 54 hours post-fertilization (hpf). Axons from at least 11 embryos from each group were transected; representative images from aSyn-expressing animals are shown. Time stamps in images are relative to the start of the imaging period. Axonal varicosities were observed (white arrowheads) several hours before cell death. White arrows point to morphological changes indicative of cell death. Inset represents cell body magnified 2×. Asterisk in A indicates separation of the axon from the cell body. Axonal fragmentation (blue arrowheads) usually did not occur before cell death, and was not stereotyped: it did not occur synchronously along the length of the axon, nor in a retrograde direction (yellow arrows point to distal portions of the axon that are still intact). (C,D) Representative images of wild-type (C) and aSyn-expressing (D) axons undergoing WD after transection with a two-photon laser. Axons were transected with a two-photon laser at 2 dpf, and embryos were imaged every 30 minutes for up to 12 hours. Red arrowhead points to site of transection. After injury, in both wild-type and aSyn-expressing axons, fragmentation was synchronous along the length of the transected axon (blue arrowheads). mpa, minutes post-axotomy. (E) There was no difference in the duration of the lag period between transection and fragmentation (WT: 129.1±10.0 minutes, n=11 axons from 11 animals; aSyn: 112.7±11.7 minutes; n=15 axons from 15 animals, P=0.3173). (F) The time between fragmentation and clearance of all axonal debris was not significantly different between the two groups (WT: 58.2±6.9 minutes; aSyn: 69.1±8.3 minutes; P=0.3213). Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4007408&req=5

f3-0070571: Axonopathy is not followed by ‘dying back’ or Wallerian-like degeneration in aSyn-expressing neurons. (A,B) Time-lapse imaging of neurodegeneration. Cells were imaged every 20 minutes beginning 54 hours post-fertilization (hpf). Axons from at least 11 embryos from each group were transected; representative images from aSyn-expressing animals are shown. Time stamps in images are relative to the start of the imaging period. Axonal varicosities were observed (white arrowheads) several hours before cell death. White arrows point to morphological changes indicative of cell death. Inset represents cell body magnified 2×. Asterisk in A indicates separation of the axon from the cell body. Axonal fragmentation (blue arrowheads) usually did not occur before cell death, and was not stereotyped: it did not occur synchronously along the length of the axon, nor in a retrograde direction (yellow arrows point to distal portions of the axon that are still intact). (C,D) Representative images of wild-type (C) and aSyn-expressing (D) axons undergoing WD after transection with a two-photon laser. Axons were transected with a two-photon laser at 2 dpf, and embryos were imaged every 30 minutes for up to 12 hours. Red arrowhead points to site of transection. After injury, in both wild-type and aSyn-expressing axons, fragmentation was synchronous along the length of the transected axon (blue arrowheads). mpa, minutes post-axotomy. (E) There was no difference in the duration of the lag period between transection and fragmentation (WT: 129.1±10.0 minutes, n=11 axons from 11 animals; aSyn: 112.7±11.7 minutes; n=15 axons from 15 animals, P=0.3173). (F) The time between fragmentation and clearance of all axonal debris was not significantly different between the two groups (WT: 58.2±6.9 minutes; aSyn: 69.1±8.3 minutes; P=0.3213). Scale bars: 50 μm.
Mentions: It has recently been proposed that the axon degeneration observed in PD represents an early, and potentially independent, process in pathophysiology (O’Malley, 2010; Burke and O’Malley, 2012; Jellinger, 2012). In zebrafish neurons expressing aSyn, the percentage of cells with dystrophic axons between 2 and 3 dpf was higher than the percentage of cells that died during that period. To determine whether severe axonopathy always preceded cell death, we conducted time-lapse imaging at 20-minute intervals between 56 and 68 hpf (Fig. 3A,B). In cells that died during the imaging period, the onset of axonal dystrophy (beading or fragmentation) was compared with morphological changes in the soma that herald cell death. In all cases (n=9), focal or diffuse swellings (axonopathy stage 2-3) were seen in axons several hours before cell death (Fig. 3A,B). Axonal fragmentation, however, did not precede apoptotic changes in the cell body (Fig. 3A,B, arrows). Overt axonal breakdown therefore does not proceed directly to the death of the cell body in this model. However, because axonal dystrophy preceded cell death, it is likely that the axonal compartment is more vulnerable to aSyn toxicity.

Bottom Line: With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system.The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection.Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.

ABSTRACT
α-synuclein (aSyn) expression is implicated in neurodegenerative processes, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In animal models of these diseases, axon pathology often precedes cell death, raising the question of whether aSyn has compartment-specific toxic effects that could require early and/or independent therapeutic intervention. The relevance of axonal pathology to degeneration can only be addressed through longitudinal, in vivo monitoring of different neuronal compartments. With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system. We therefore expressed human wild-type aSyn in zebrafish peripheral sensory neurons, which project elaborate superficial axons that can be continuously imaged in vivo. Axonal outgrowth was normal in these neurons but, by 2 days post-fertilization (dpf), many aSyn-expressing axons became dystrophic, with focal varicosities or diffuse beading. Approximately 20% of aSyn-expressing cells died by 3 dpf. Time-lapse imaging revealed that focal axonal swelling, but not overt fragmentation, usually preceded cell death. Co-expressing aSyn with a mitochondrial reporter revealed deficits in mitochondrial transport and morphology even when axons appeared overtly normal. The axon-protective protein Wallerian degeneration slow (WldS) delayed axon degeneration but not cell death caused by aSyn. By contrast, the transcriptional coactivator PGC-1α, which has roles in the regulation of mitochondrial biogenesis and reactive-oxygen-species detoxification, abrogated aSyn toxicity in both the axon and the cell body. The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection. Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds.

Show MeSH
Related in: MedlinePlus