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Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults

View Article: PubMed Central - PubMed

ABSTRACT

In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson’s and Alzheimer’s, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events.

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Genetic manipulation of mitochondrial dynamics promotes retrograde spreading of axonal degeneration toward the cell body after local rotenone insult.(a–f) Representative fluorescence images (mosaic tiled reconstruction into one image) of neurons from CGN, 10 days after co-transfection with control, OPA1, OPA1G300E or DRP1 and GFP vectors seeded in microfluidic chambers. CGN were grown in high glucose (HG) condition and axons were treated with vehicle (Control) (a,c,e) or 5 μM rotenone (b,d,f) for 24 hours. Note the formation of axonal blebbing reminiscent of axonal degeneration inside the micro-channel area. (g,h) quantification of the axonal degeneration index in the distal (g) and proximal parts (h) of the micro-channel areas. Each experiment was conducted at least 3 times independently in triplicates and data were analyzed using ANOVA statistical method.
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f4: Genetic manipulation of mitochondrial dynamics promotes retrograde spreading of axonal degeneration toward the cell body after local rotenone insult.(a–f) Representative fluorescence images (mosaic tiled reconstruction into one image) of neurons from CGN, 10 days after co-transfection with control, OPA1, OPA1G300E or DRP1 and GFP vectors seeded in microfluidic chambers. CGN were grown in high glucose (HG) condition and axons were treated with vehicle (Control) (a,c,e) or 5 μM rotenone (b,d,f) for 24 hours. Note the formation of axonal blebbing reminiscent of axonal degeneration inside the micro-channel area. (g,h) quantification of the axonal degeneration index in the distal (g) and proximal parts (h) of the micro-channel areas. Each experiment was conducted at least 3 times independently in triplicates and data were analyzed using ANOVA statistical method.

Mentions: As described in Fig. 2, axonal application of rotenone triggers destruction of the treated axonal endings associated with a minor and very slow retrograde degeneration profile inside the micro-channel area. A plausible scenario would be that mitochondria lying in the unchallenged parts of the axons may act as buffer, limiting the retrograde spread of locally imitated insults. In order to assess whether mitochondrial fission may modify retrograde degeneration, together with OPA1G300E or DRP1 plasmids CGN neurons were co-transfected with a GFP encoding vector allowing visualization of the complete neuronal morphology. DIV10 GCN, grown in HG conditions, were treated with rotenone applied on the axonal chamber. Morphological integrity of both distal and proximal parts of the axons individualized in the micro-channels was then recorded. As shown in Fig. 4a,b, under these high glucose culture conditions, rotenone treatment did not result in a significant axonal degeneration in control CGN, with very few fragmentation events observed only inside the distal part of the micro-channels. Strikingly, axons from neurons transfected with OPA1G300E (Fig. 4c,d) and DRP1 (Fig. 4e,f) and treated with rotenone, show extensive signs of degeneration in the distal parts of the micro-channel. In DRP1-overexpressing neurons, and to a lesser extent for OPA1G300E expressing neurons, this is associated with further signs of retrograde degeneration toward the proximal part of the micro-channels (Fig. 4c–f) and somatic degeneration in the somatic chamber (not shown). Quantification of the axonal degeneration index in the micro-channels clearly indicates that both DRP1 and OPA1G300E promote a fast retrograde degeneration process (Fig. 4g,h).


Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults
Genetic manipulation of mitochondrial dynamics promotes retrograde spreading of axonal degeneration toward the cell body after local rotenone insult.(a–f) Representative fluorescence images (mosaic tiled reconstruction into one image) of neurons from CGN, 10 days after co-transfection with control, OPA1, OPA1G300E or DRP1 and GFP vectors seeded in microfluidic chambers. CGN were grown in high glucose (HG) condition and axons were treated with vehicle (Control) (a,c,e) or 5 μM rotenone (b,d,f) for 24 hours. Note the formation of axonal blebbing reminiscent of axonal degeneration inside the micro-channel area. (g,h) quantification of the axonal degeneration index in the distal (g) and proximal parts (h) of the micro-channel areas. Each experiment was conducted at least 3 times independently in triplicates and data were analyzed using ANOVA statistical method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Genetic manipulation of mitochondrial dynamics promotes retrograde spreading of axonal degeneration toward the cell body after local rotenone insult.(a–f) Representative fluorescence images (mosaic tiled reconstruction into one image) of neurons from CGN, 10 days after co-transfection with control, OPA1, OPA1G300E or DRP1 and GFP vectors seeded in microfluidic chambers. CGN were grown in high glucose (HG) condition and axons were treated with vehicle (Control) (a,c,e) or 5 μM rotenone (b,d,f) for 24 hours. Note the formation of axonal blebbing reminiscent of axonal degeneration inside the micro-channel area. (g,h) quantification of the axonal degeneration index in the distal (g) and proximal parts (h) of the micro-channel areas. Each experiment was conducted at least 3 times independently in triplicates and data were analyzed using ANOVA statistical method.
Mentions: As described in Fig. 2, axonal application of rotenone triggers destruction of the treated axonal endings associated with a minor and very slow retrograde degeneration profile inside the micro-channel area. A plausible scenario would be that mitochondria lying in the unchallenged parts of the axons may act as buffer, limiting the retrograde spread of locally imitated insults. In order to assess whether mitochondrial fission may modify retrograde degeneration, together with OPA1G300E or DRP1 plasmids CGN neurons were co-transfected with a GFP encoding vector allowing visualization of the complete neuronal morphology. DIV10 GCN, grown in HG conditions, were treated with rotenone applied on the axonal chamber. Morphological integrity of both distal and proximal parts of the axons individualized in the micro-channels was then recorded. As shown in Fig. 4a,b, under these high glucose culture conditions, rotenone treatment did not result in a significant axonal degeneration in control CGN, with very few fragmentation events observed only inside the distal part of the micro-channels. Strikingly, axons from neurons transfected with OPA1G300E (Fig. 4c,d) and DRP1 (Fig. 4e,f) and treated with rotenone, show extensive signs of degeneration in the distal parts of the micro-channel. In DRP1-overexpressing neurons, and to a lesser extent for OPA1G300E expressing neurons, this is associated with further signs of retrograde degeneration toward the proximal part of the micro-channels (Fig. 4c–f) and somatic degeneration in the somatic chamber (not shown). Quantification of the axonal degeneration index in the micro-channels clearly indicates that both DRP1 and OPA1G300E promote a fast retrograde degeneration process (Fig. 4g,h).

View Article: PubMed Central - PubMed

ABSTRACT

In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson’s and Alzheimer’s, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events.

No MeSH data available.


Related in: MedlinePlus