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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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Early mitochondrial pathology and transport impairments in aSyn-expressing axons. (A) Transgenes used to visualize mitochondria in GFP-(WT) or aSyn-2A-GFP-expressing peripheral sensory neurons. Transgenes were co-injected into wild-type embryos at the one-cell stage. WT (B-B″) and aSyn-expressing (C-C″) cells were imaged at 2 dpf. Yellow arrowheads point to elongated mitochondria in wild-type axons. (D) Mitochondrial density was higher in aSyn-expressing cells (WT: 226.2±19.1 mitochondria/μm, n=26 axons in 12 animals; aSyn: 302.0±29.8 mitochondria/μm; n=20 axons in 10 animals, *P =0.031). (E) Mitochondrial morphology was quantified as the ratio of length to width in individual mitochondria. Values were binned and the frequency distribution was plotted on a histogram. Mitochondria in aSyn-expressing axons were more spherical than in wild-type axons, with fewer mitochondria exhibiting a high length:width ratio. (F) Large, swollen mitochondria occupied the spheroids in dystrophic aSyn-expressing axons. Boxed region in F is represented in F′-F″. Red arrowheads point to enlarged mitochondria. [Note that the scale bar in F″ is the same as in B″ and C″ (100 μm).] (G) Mitochondrial transport was evaluated along 50-μm axonal segments every second. Overall mitochondrial transport was significantly reduced in aSyn-expressing axons (WT % motile: 27.4±2.7%; aSyn: 15.05±3.4%; n≥20 axons in ≥10 animals per group, as above; **P =0.0061). (H) A higher percentage of distance traveled by motile mitochondria was in the retrograde direction (WT % retrograde distance: 44.61±5.07%; aSyn: 62.17±6.06%; n≥52 mitochondria; *P =0.0300). (I) Motile mitochondria spent less time moving in the anterograde direction (WT: 36.44±4.80%; aSyn: 17.39±4.05%; n≥52 mitochondria; **P=0.0063), and a greater percentage of time paused than in wild-type axons (WT: 41.25±4.43%; aSyn: 54.82±4.85%; n≥52 mitochondria; *P=0.0478).
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f5-0070571: Early mitochondrial pathology and transport impairments in aSyn-expressing axons. (A) Transgenes used to visualize mitochondria in GFP-(WT) or aSyn-2A-GFP-expressing peripheral sensory neurons. Transgenes were co-injected into wild-type embryos at the one-cell stage. WT (B-B″) and aSyn-expressing (C-C″) cells were imaged at 2 dpf. Yellow arrowheads point to elongated mitochondria in wild-type axons. (D) Mitochondrial density was higher in aSyn-expressing cells (WT: 226.2±19.1 mitochondria/μm, n=26 axons in 12 animals; aSyn: 302.0±29.8 mitochondria/μm; n=20 axons in 10 animals, *P =0.031). (E) Mitochondrial morphology was quantified as the ratio of length to width in individual mitochondria. Values were binned and the frequency distribution was plotted on a histogram. Mitochondria in aSyn-expressing axons were more spherical than in wild-type axons, with fewer mitochondria exhibiting a high length:width ratio. (F) Large, swollen mitochondria occupied the spheroids in dystrophic aSyn-expressing axons. Boxed region in F is represented in F′-F″. Red arrowheads point to enlarged mitochondria. [Note that the scale bar in F″ is the same as in B″ and C″ (100 μm).] (G) Mitochondrial transport was evaluated along 50-μm axonal segments every second. Overall mitochondrial transport was significantly reduced in aSyn-expressing axons (WT % motile: 27.4±2.7%; aSyn: 15.05±3.4%; n≥20 axons in ≥10 animals per group, as above; **P =0.0061). (H) A higher percentage of distance traveled by motile mitochondria was in the retrograde direction (WT % retrograde distance: 44.61±5.07%; aSyn: 62.17±6.06%; n≥52 mitochondria; *P =0.0300). (I) Motile mitochondria spent less time moving in the anterograde direction (WT: 36.44±4.80%; aSyn: 17.39±4.05%; n≥52 mitochondria; **P=0.0063), and a greater percentage of time paused than in wild-type axons (WT: 41.25±4.43%; aSyn: 54.82±4.85%; n≥52 mitochondria; *P=0.0478).

Mentions: Multiple in vitro and histological studies suggest that both wild-type and mutant aSyn interact with mitochondria (Martin et al., 2006; Parihar et al., 2008; Banerjee et al., 2010; Chinta et al., 2010; Devi and Anandatheerthavarada, 2010; Nakamura et al., 2011; Calì et al., 2012; Reeve et al., 2012; Zhu et al., 2012). To determine whether axonal mitochondria were affected by aSyn expression in our model, DsRed fused to the cox8 mitochondrial matrix targeting signal was co-expressed in sensory neurons with either GFP or aSyn-2A-GFP (Fig. 5A–C). Mitochondrial density was significantly higher in aSyn-expressing cells, even in the absence of overt axonopathy (Fig. 5C,D). Mitochondria in aSyn-expressing axons were less elongated than in wild-type cells (Fig. 5C,E; WT length/width: 2.01±0.11; aSyn: 1.48±0.05; n≥54 mitochondria from ≥5 embryos; P<0.0001), with a higher percentage of spherical mitochondria (ratio of 1), a phenotype associated with respiratory chain dysfunction (Benard and Rossignol, 2008). In dystrophic aSyn-expressing axons (Fig. 5F), many mitochondria exhibited pathological swelling characteristic of the mitochondrial permeability transition (Haworth and Hunter, 1979; Kowaltowski et al., 1996; Brustovetsky et al., 2002).


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)

Early mitochondrial pathology and transport impairments in aSyn-expressing axons. (A) Transgenes used to visualize mitochondria in GFP-(WT) or aSyn-2A-GFP-expressing peripheral sensory neurons. Transgenes were co-injected into wild-type embryos at the one-cell stage. WT (B-B″) and aSyn-expressing (C-C″) cells were imaged at 2 dpf. Yellow arrowheads point to elongated mitochondria in wild-type axons. (D) Mitochondrial density was higher in aSyn-expressing cells (WT: 226.2±19.1 mitochondria/μm, n=26 axons in 12 animals; aSyn: 302.0±29.8 mitochondria/μm; n=20 axons in 10 animals, *P =0.031). (E) Mitochondrial morphology was quantified as the ratio of length to width in individual mitochondria. Values were binned and the frequency distribution was plotted on a histogram. Mitochondria in aSyn-expressing axons were more spherical than in wild-type axons, with fewer mitochondria exhibiting a high length:width ratio. (F) Large, swollen mitochondria occupied the spheroids in dystrophic aSyn-expressing axons. Boxed region in F is represented in F′-F″. Red arrowheads point to enlarged mitochondria. [Note that the scale bar in F″ is the same as in B″ and C″ (100 μm).] (G) Mitochondrial transport was evaluated along 50-μm axonal segments every second. Overall mitochondrial transport was significantly reduced in aSyn-expressing axons (WT % motile: 27.4±2.7%; aSyn: 15.05±3.4%; n≥20 axons in ≥10 animals per group, as above; **P =0.0061). (H) A higher percentage of distance traveled by motile mitochondria was in the retrograde direction (WT % retrograde distance: 44.61±5.07%; aSyn: 62.17±6.06%; n≥52 mitochondria; *P =0.0300). (I) Motile mitochondria spent less time moving in the anterograde direction (WT: 36.44±4.80%; aSyn: 17.39±4.05%; n≥52 mitochondria; **P=0.0063), and a greater percentage of time paused than in wild-type axons (WT: 41.25±4.43%; aSyn: 54.82±4.85%; n≥52 mitochondria; *P=0.0478).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-0070571: Early mitochondrial pathology and transport impairments in aSyn-expressing axons. (A) Transgenes used to visualize mitochondria in GFP-(WT) or aSyn-2A-GFP-expressing peripheral sensory neurons. Transgenes were co-injected into wild-type embryos at the one-cell stage. WT (B-B″) and aSyn-expressing (C-C″) cells were imaged at 2 dpf. Yellow arrowheads point to elongated mitochondria in wild-type axons. (D) Mitochondrial density was higher in aSyn-expressing cells (WT: 226.2±19.1 mitochondria/μm, n=26 axons in 12 animals; aSyn: 302.0±29.8 mitochondria/μm; n=20 axons in 10 animals, *P =0.031). (E) Mitochondrial morphology was quantified as the ratio of length to width in individual mitochondria. Values were binned and the frequency distribution was plotted on a histogram. Mitochondria in aSyn-expressing axons were more spherical than in wild-type axons, with fewer mitochondria exhibiting a high length:width ratio. (F) Large, swollen mitochondria occupied the spheroids in dystrophic aSyn-expressing axons. Boxed region in F is represented in F′-F″. Red arrowheads point to enlarged mitochondria. [Note that the scale bar in F″ is the same as in B″ and C″ (100 μm).] (G) Mitochondrial transport was evaluated along 50-μm axonal segments every second. Overall mitochondrial transport was significantly reduced in aSyn-expressing axons (WT % motile: 27.4±2.7%; aSyn: 15.05±3.4%; n≥20 axons in ≥10 animals per group, as above; **P =0.0061). (H) A higher percentage of distance traveled by motile mitochondria was in the retrograde direction (WT % retrograde distance: 44.61±5.07%; aSyn: 62.17±6.06%; n≥52 mitochondria; *P =0.0300). (I) Motile mitochondria spent less time moving in the anterograde direction (WT: 36.44±4.80%; aSyn: 17.39±4.05%; n≥52 mitochondria; **P=0.0063), and a greater percentage of time paused than in wild-type axons (WT: 41.25±4.43%; aSyn: 54.82±4.85%; n≥52 mitochondria; *P=0.0478).
Mentions: Multiple in vitro and histological studies suggest that both wild-type and mutant aSyn interact with mitochondria (Martin et al., 2006; Parihar et al., 2008; Banerjee et al., 2010; Chinta et al., 2010; Devi and Anandatheerthavarada, 2010; Nakamura et al., 2011; Calì et al., 2012; Reeve et al., 2012; Zhu et al., 2012). To determine whether axonal mitochondria were affected by aSyn expression in our model, DsRed fused to the cox8 mitochondrial matrix targeting signal was co-expressed in sensory neurons with either GFP or aSyn-2A-GFP (Fig. 5A–C). Mitochondrial density was significantly higher in aSyn-expressing cells, even in the absence of overt axonopathy (Fig. 5C,D). Mitochondria in aSyn-expressing axons were less elongated than in wild-type cells (Fig. 5C,E; WT length/width: 2.01±0.11; aSyn: 1.48±0.05; n≥54 mitochondria from ≥5 embryos; P<0.0001), with a higher percentage of spherical mitochondria (ratio of 1), a phenotype associated with respiratory chain dysfunction (Benard and Rossignol, 2008). In dystrophic aSyn-expressing axons (Fig. 5F), many mitochondria exhibited pathological swelling characteristic of the mitochondrial permeability transition (Haworth and Hunter, 1979; Kowaltowski et al., 1996; Brustovetsky et al., 2002).

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