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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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Local pharmacological inhibition of mitochondrial fission prevented axonal damage.(a–l) Paired morphology panel of DIV10 CGN in microfluidic chambers. The somatic chamber was probed with Hoechst nuclear staining (a,c,e,g,i,k) and axonal endings in the axonal chamber immuno-stained with beta3-tubulin (b,d,f,h,j,l). (a,b), 10 DIV CGN grown in high glucose (HG) conditions. (c,d) Application of axonal rotenone (Rot HG) in the axonal chamber did not trigger axonal degeneration in HG condition after 24 hours of treatment. (e,f) Low glucose (LG) condition was innocuous. (g,h) Application of axonal rotenone in LG triggered axonal degeneration. (i,j) Application of 10 μM Mdivi-1 on axons inhibited the action of rotenone, but 10 μM SP600125 did not (k,l). (Scale bar: 10 μm). (m) Quantification of axonal degeneration. Each experiment was conducted at 3 times independently in triplicates and data were analyzed using ANOVA statistical method. (n) Representative images of both proximal (somatic chambers) and distal (axonal chamber) axonal mitochondrial morphology upon TOM20 immunostaining after rotenone insult and pharmacological blockade.
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f2: Local pharmacological inhibition of mitochondrial fission prevented axonal damage.(a–l) Paired morphology panel of DIV10 CGN in microfluidic chambers. The somatic chamber was probed with Hoechst nuclear staining (a,c,e,g,i,k) and axonal endings in the axonal chamber immuno-stained with beta3-tubulin (b,d,f,h,j,l). (a,b), 10 DIV CGN grown in high glucose (HG) conditions. (c,d) Application of axonal rotenone (Rot HG) in the axonal chamber did not trigger axonal degeneration in HG condition after 24 hours of treatment. (e,f) Low glucose (LG) condition was innocuous. (g,h) Application of axonal rotenone in LG triggered axonal degeneration. (i,j) Application of 10 μM Mdivi-1 on axons inhibited the action of rotenone, but 10 μM SP600125 did not (k,l). (Scale bar: 10 μm). (m) Quantification of axonal degeneration. Each experiment was conducted at 3 times independently in triplicates and data were analyzed using ANOVA statistical method. (n) Representative images of both proximal (somatic chambers) and distal (axonal chamber) axonal mitochondrial morphology upon TOM20 immunostaining after rotenone insult and pharmacological blockade.

Mentions: While increasing mitochondrial fission per se did not lead to spontaneous axonal degeneration, mitochondrial fission could sensitize axons toward stressors such as rotenone. This mitochondrial poison promotes neuronal apoptosis and primarily targets the Complex I of the mitochondrial respiratory chain, triggers mitochondrial ROS production. Due to microfluidic barriers and axons occluding the micro channels, molecular diffusion between axonal and somatic chambers is highly limited4243. Using this to our advantage, we tested whether mitochondrial fission modifies the vulnerability of axons toward susceptibility to pro apoptotic stressor. Rotenone was selectively applied to CGN axonal compartments in order to initiate local degenerative events. Axonal application of up to 5 μM of rotenone in High Glucose (HG) condition had no visible effect on both axonal mitochondrial morphology and axonal degeneration (Fig. 2a–d,m,n). However, as previously observed13, in glycolytically impaired conditions (i.e. in Low Glucose (LG) conditions), rotenone induced extensive axonal mitochondrial fission and axonal degeneration (Fig. 2e–h,m,n). A striking observation was that while rotenone treatment of axonal endings in glycolytically impaired conditions triggers complete axonal degeneration in the distal (treated) chambers, there is almost no observable retrograde spreading of the axonal damage toward the cell body, as assessed by nuclear integrity staining after 24 hours (Fig. 2g). Yet, careful examination and morphological analysis of GFP-transfected at a later time point (axons treated for 48 hours) showed evidence of a slow and partial retrograde degeneration (Sup Fig. 1).


Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults
Local pharmacological inhibition of mitochondrial fission prevented axonal damage.(a–l) Paired morphology panel of DIV10 CGN in microfluidic chambers. The somatic chamber was probed with Hoechst nuclear staining (a,c,e,g,i,k) and axonal endings in the axonal chamber immuno-stained with beta3-tubulin (b,d,f,h,j,l). (a,b), 10 DIV CGN grown in high glucose (HG) conditions. (c,d) Application of axonal rotenone (Rot HG) in the axonal chamber did not trigger axonal degeneration in HG condition after 24 hours of treatment. (e,f) Low glucose (LG) condition was innocuous. (g,h) Application of axonal rotenone in LG triggered axonal degeneration. (i,j) Application of 10 μM Mdivi-1 on axons inhibited the action of rotenone, but 10 μM SP600125 did not (k,l). (Scale bar: 10 μm). (m) Quantification of axonal degeneration. Each experiment was conducted at 3 times independently in triplicates and data were analyzed using ANOVA statistical method. (n) Representative images of both proximal (somatic chambers) and distal (axonal chamber) axonal mitochondrial morphology upon TOM20 immunostaining after rotenone insult and pharmacological blockade.
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Related In: Results  -  Collection

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f2: Local pharmacological inhibition of mitochondrial fission prevented axonal damage.(a–l) Paired morphology panel of DIV10 CGN in microfluidic chambers. The somatic chamber was probed with Hoechst nuclear staining (a,c,e,g,i,k) and axonal endings in the axonal chamber immuno-stained with beta3-tubulin (b,d,f,h,j,l). (a,b), 10 DIV CGN grown in high glucose (HG) conditions. (c,d) Application of axonal rotenone (Rot HG) in the axonal chamber did not trigger axonal degeneration in HG condition after 24 hours of treatment. (e,f) Low glucose (LG) condition was innocuous. (g,h) Application of axonal rotenone in LG triggered axonal degeneration. (i,j) Application of 10 μM Mdivi-1 on axons inhibited the action of rotenone, but 10 μM SP600125 did not (k,l). (Scale bar: 10 μm). (m) Quantification of axonal degeneration. Each experiment was conducted at 3 times independently in triplicates and data were analyzed using ANOVA statistical method. (n) Representative images of both proximal (somatic chambers) and distal (axonal chamber) axonal mitochondrial morphology upon TOM20 immunostaining after rotenone insult and pharmacological blockade.
Mentions: While increasing mitochondrial fission per se did not lead to spontaneous axonal degeneration, mitochondrial fission could sensitize axons toward stressors such as rotenone. This mitochondrial poison promotes neuronal apoptosis and primarily targets the Complex I of the mitochondrial respiratory chain, triggers mitochondrial ROS production. Due to microfluidic barriers and axons occluding the micro channels, molecular diffusion between axonal and somatic chambers is highly limited4243. Using this to our advantage, we tested whether mitochondrial fission modifies the vulnerability of axons toward susceptibility to pro apoptotic stressor. Rotenone was selectively applied to CGN axonal compartments in order to initiate local degenerative events. Axonal application of up to 5 μM of rotenone in High Glucose (HG) condition had no visible effect on both axonal mitochondrial morphology and axonal degeneration (Fig. 2a–d,m,n). However, as previously observed13, in glycolytically impaired conditions (i.e. in Low Glucose (LG) conditions), rotenone induced extensive axonal mitochondrial fission and axonal degeneration (Fig. 2e–h,m,n). A striking observation was that while rotenone treatment of axonal endings in glycolytically impaired conditions triggers complete axonal degeneration in the distal (treated) chambers, there is almost no observable retrograde spreading of the axonal damage toward the cell body, as assessed by nuclear integrity staining after 24 hours (Fig. 2g). Yet, careful examination and morphological analysis of GFP-transfected at a later time point (axons treated for 48 hours) showed evidence of a slow and partial retrograde degeneration (Sup Fig. 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