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

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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 affects axonal vulnerability to rotenone.(a–p) Representative fluorescence images of axons from CGN, 10 days after co-transfection with 1) MitoDsRed vectors and 2) control, OPA1, OPA1G300E or DRP1 vectors. Axonal endings were visualized after β3-tubulin immunostaining (left panels) and mitochondria by Mito-DsRed fluorescence (right panels). CGN were grown in HG condition and axons were treated with vehicle (Control) (a,b,e,f,i,j,m,n) or 5 μM rotenone (c,d,g,h,k,l,o,p) for 24 hours. (q) Bar graph of the quantification of axonal degeneration in all conditions. Each experiment was conducted 3 times independently in triplicates and data were analyzed using ANOVA statistical method. Insert: Representative images of axonal mitochondria upon the various conditions studied.
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f3: Genetic manipulation of mitochondrial dynamics affects axonal vulnerability to rotenone.(a–p) Representative fluorescence images of axons from CGN, 10 days after co-transfection with 1) MitoDsRed vectors and 2) control, OPA1, OPA1G300E or DRP1 vectors. Axonal endings were visualized after β3-tubulin immunostaining (left panels) and mitochondria by Mito-DsRed fluorescence (right panels). CGN were grown in HG condition and axons were treated with vehicle (Control) (a,b,e,f,i,j,m,n) or 5 μM rotenone (c,d,g,h,k,l,o,p) for 24 hours. (q) Bar graph of the quantification of axonal degeneration in all conditions. Each experiment was conducted 3 times independently in triplicates and data were analyzed using ANOVA statistical method. Insert: Representative images of axonal mitochondria upon the various conditions studied.

Mentions: Alteration of mitochondrial dynamics by genetic means may lead to subtle axonal alterations in mitochondrial physiology, which although non-lethal per se, have been proposed to sensitize cells to further stress2841. In order to assess whether fragmented axonal mitochondria are associated with increased vulnerability to axonal rotenone, we exposed the axons of OPA1-, OPA1G300E- and DRP1-transfected neurons to 5 μM rotenone in HG cell culture medium, a sub-threshold condition in which rotenone has no deleterious effect. As shown in Fig. 3a–d,q, in HG medium, the expression of control and Mito-DsRed vectors, alone or in combination with rotenone (5 μM) axonal application, had no effect on mitochondrial morphology or axonal integrity. Overexpression of OPA1 did not modify axonal integrity or the overall mitochondrial morphology with or without rotenone (Fig. 3e–h). However, application of rotenone in HG medium of both OPA1G300E (Fig. 3i–l) and DRP1 (Fig. 3m–p) treated axons resulted in 30% axonal fragmentation after a 24 h treatment (Fig. 3q). It can be concluded that mitochondrial fission significantly enhances axonal vulnerability to rotenone and allows to overcome the inhibitory effect of high glycolytic environment. Interestingly, at time points where axons show no sign of degeneration (6h), rotenone treatment of axonal segment triggered a decrease in both mitochondria movement (from 33% to 12% ) and speed (0.95 to 0.04 μm/s−1). This effect was even more pronounced under DRP1 overexpression condition where mitochondria virtually halted in the treated segments (motility drops from 30% to 1.8% and speed from 0.05 to 0.015 μm/s−1).


Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults
Genetic manipulation of mitochondrial dynamics affects axonal vulnerability to rotenone.(a–p) Representative fluorescence images of axons from CGN, 10 days after co-transfection with 1) MitoDsRed vectors and 2) control, OPA1, OPA1G300E or DRP1 vectors. Axonal endings were visualized after β3-tubulin immunostaining (left panels) and mitochondria by Mito-DsRed fluorescence (right panels). CGN were grown in HG condition and axons were treated with vehicle (Control) (a,b,e,f,i,j,m,n) or 5 μM rotenone (c,d,g,h,k,l,o,p) for 24 hours. (q) Bar graph of the quantification of axonal degeneration in all conditions. Each experiment was conducted 3 times independently in triplicates and data were analyzed using ANOVA statistical method. Insert: Representative images of axonal mitochondria upon the various conditions studied.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5015069&req=5

f3: Genetic manipulation of mitochondrial dynamics affects axonal vulnerability to rotenone.(a–p) Representative fluorescence images of axons from CGN, 10 days after co-transfection with 1) MitoDsRed vectors and 2) control, OPA1, OPA1G300E or DRP1 vectors. Axonal endings were visualized after β3-tubulin immunostaining (left panels) and mitochondria by Mito-DsRed fluorescence (right panels). CGN were grown in HG condition and axons were treated with vehicle (Control) (a,b,e,f,i,j,m,n) or 5 μM rotenone (c,d,g,h,k,l,o,p) for 24 hours. (q) Bar graph of the quantification of axonal degeneration in all conditions. Each experiment was conducted 3 times independently in triplicates and data were analyzed using ANOVA statistical method. Insert: Representative images of axonal mitochondria upon the various conditions studied.
Mentions: Alteration of mitochondrial dynamics by genetic means may lead to subtle axonal alterations in mitochondrial physiology, which although non-lethal per se, have been proposed to sensitize cells to further stress2841. In order to assess whether fragmented axonal mitochondria are associated with increased vulnerability to axonal rotenone, we exposed the axons of OPA1-, OPA1G300E- and DRP1-transfected neurons to 5 μM rotenone in HG cell culture medium, a sub-threshold condition in which rotenone has no deleterious effect. As shown in Fig. 3a–d,q, in HG medium, the expression of control and Mito-DsRed vectors, alone or in combination with rotenone (5 μM) axonal application, had no effect on mitochondrial morphology or axonal integrity. Overexpression of OPA1 did not modify axonal integrity or the overall mitochondrial morphology with or without rotenone (Fig. 3e–h). However, application of rotenone in HG medium of both OPA1G300E (Fig. 3i–l) and DRP1 (Fig. 3m–p) treated axons resulted in 30% axonal fragmentation after a 24 h treatment (Fig. 3q). It can be concluded that mitochondrial fission significantly enhances axonal vulnerability to rotenone and allows to overcome the inhibitory effect of high glycolytic environment. Interestingly, at time points where axons show no sign of degeneration (6h), rotenone treatment of axonal segment triggered a decrease in both mitochondria movement (from 33% to 12% ) and speed (0.95 to 0.04 μm/s−1). This effect was even more pronounced under DRP1 overexpression condition where mitochondria virtually halted in the treated segments (motility drops from 30% to 1.8% and speed from 0.05 to 0.015 μm/s−1).

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