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Autophagy-mediated turnover of dynamin-related protein 1.

Purnell PR, Fox HS - BMC Neurosci (2013)

Bottom Line: We are able to decrease Drp1 levels in a time- and dose-dependent manner with the potent neuronal autophagy inducer 10-NCP, as well as structurally related compounds.Further, 10-NCP was able increase average mitochondrial size and length verifying a functional result of Drp1 depletion in these neurons.Additionally these data suggest a mechanism, through Drp1 downregulation, which may partly explain the ability of autophagy to have a neuroprotective effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT

Background: Drp1 is the primary protein responsible for mitochondrial fission. Perturbations of mitochondrial morphology and increased fission are seen in neurodegeneration. While Drp1 degradation induced by Parkin overexpression can be prevented by proteasome inhibition, there are numerous links between proteasomal and autophagic processes in mitochondrial protein degradation. Here we investigated the role of autophagy in Drp1 regulation.

Results: We demonstrate that autophagy plays a major role in the control of Drp1 levels. In HEK-293T cells, inhibitors of autophagy increase total Drp1 and levels of Drp1 in the mitochondrial cellular fraction. Similarly by silencing ATG7, which is required for initiation of autophagy, there is an increased level of Drp1. Because of the role of increased Drp1 in neurodegeneration, we then examined the ability to modulate Drp1 levels in neurons by inducing autophagy. We are able to decrease Drp1 levels in a time- and dose-dependent manner with the potent neuronal autophagy inducer 10-NCP, as well as structurally related compounds. Further, 10-NCP was able increase average mitochondrial size and length verifying a functional result of Drp1 depletion in these neurons.

Conclusions: These pharmacological and genetic approaches indicate that autophagy targets Drp1 for lysosomal degradation. Additionally these data suggest a mechanism, through Drp1 downregulation, which may partly explain the ability of autophagy to have a neuroprotective effect.

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Inducing autophagy decreases Drp1 levels in rat-derived striatal neurons. (A) E18 rat-derived striatal neurons were incubated with 10-NCP at the concentrations indicated for 12 hours. (B) Neurons were incubated with 5 μM 10-NCP for the indicated times. (C and D) Chlorpromazine and Trifluoperazine (5 μM) was incubated for the times indicated followed by Western blotting. (E) Striatal neurons were incubated with 10-NCP for 12 hours along with transduction of CellLight Mitochondria GFP to allow labeling and visualization of mitochondria. (F) Frequency distribution of mitochondrial size in control and 10-NCP treated neurons as quantitated from confocal immunofluorescent images using the Mitochondrial Morphology plug-in for ImageJ (n > 500 for each condition, X-axis truncated at 200). (G and H) Box and whisker plot (with whiskers representing 5-95th percentile) of the average mitochondrial size and major axis per neuron (n = 20). *P < 0.05.
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Figure 3: Inducing autophagy decreases Drp1 levels in rat-derived striatal neurons. (A) E18 rat-derived striatal neurons were incubated with 10-NCP at the concentrations indicated for 12 hours. (B) Neurons were incubated with 5 μM 10-NCP for the indicated times. (C and D) Chlorpromazine and Trifluoperazine (5 μM) was incubated for the times indicated followed by Western blotting. (E) Striatal neurons were incubated with 10-NCP for 12 hours along with transduction of CellLight Mitochondria GFP to allow labeling and visualization of mitochondria. (F) Frequency distribution of mitochondrial size in control and 10-NCP treated neurons as quantitated from confocal immunofluorescent images using the Mitochondrial Morphology plug-in for ImageJ (n > 500 for each condition, X-axis truncated at 200). (G and H) Box and whisker plot (with whiskers representing 5-95th percentile) of the average mitochondrial size and major axis per neuron (n = 20). *P < 0.05.

Mentions: Imbalanced mitochondrial fusion and fission has been well characterized in neurodegeneration [26]. Increased Drp1 levels in AD [18] and HD patients [27], and cellular data from PD models all suggest that increased Drp1 is associated with pathogenesis. With this and evidence for autophagy-mediated turnover of Drp1 in mind, we studied the possible effect of inducing autophagy in a primary neurons. The compound 10-NCP has been found to potently induce autophagy in primary neurons [28]. Using cultured E18 rat-derived striatal neurons, 10-NCP is able to reduce Drp1 levels in a dose-dependent manner with a corresponding increase in LC3-II levels (Figure 3A). This effect is also time dependent and Drp1 is effectively decreased by 24 hours of 10-NCP treatment (Figure 3B). The previous study of 10-NCP in neurons also identified several FDA-approved structural analogs, which were able to analogously induce autophagy [28]. We were interested to see if these compounds would act similarly to 10-NCP in these neurons. Both compounds tested (chlorpromazine and trifluoperazine) were able to decrease Drp1 in a similar time dependent manner to 10-NCP (Figure 3C-D). To confirm the functional relevance of Drp1 downregulation, E18 rat-derived striatal neurons were treated with vehicle or 10-NCP along with transduction of a mitochondrially-targeted GFP (Figure 3E). It appears that 10-NCP leads to decreased fission as mitochondria become more interconnected after treatment (Figure 3E). To quantify this effect, mitochondrial morphology was analyzed using a well-characterized image analysis program (ImageJ plug-in) [22]. After 10-NCP treatments, mitochondrial size is increased in a large percentage of the neuronal mitochondria (Figure 3F). To better quantify this effect, the average and distribution of mitochondrial size and length (defined by the major mitochondrial axis) in control and 10-NCP treated neurons is shown (Figure 3G-H). These data suggest the induction of autophagy in neurons is able to decrease Drp1 and this approach may be useful to decrease the aberrant Drp1 expression observed in neurodegeneration.


Autophagy-mediated turnover of dynamin-related protein 1.

Purnell PR, Fox HS - BMC Neurosci (2013)

Inducing autophagy decreases Drp1 levels in rat-derived striatal neurons. (A) E18 rat-derived striatal neurons were incubated with 10-NCP at the concentrations indicated for 12 hours. (B) Neurons were incubated with 5 μM 10-NCP for the indicated times. (C and D) Chlorpromazine and Trifluoperazine (5 μM) was incubated for the times indicated followed by Western blotting. (E) Striatal neurons were incubated with 10-NCP for 12 hours along with transduction of CellLight Mitochondria GFP to allow labeling and visualization of mitochondria. (F) Frequency distribution of mitochondrial size in control and 10-NCP treated neurons as quantitated from confocal immunofluorescent images using the Mitochondrial Morphology plug-in for ImageJ (n > 500 for each condition, X-axis truncated at 200). (G and H) Box and whisker plot (with whiskers representing 5-95th percentile) of the average mitochondrial size and major axis per neuron (n = 20). *P < 0.05.
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Figure 3: Inducing autophagy decreases Drp1 levels in rat-derived striatal neurons. (A) E18 rat-derived striatal neurons were incubated with 10-NCP at the concentrations indicated for 12 hours. (B) Neurons were incubated with 5 μM 10-NCP for the indicated times. (C and D) Chlorpromazine and Trifluoperazine (5 μM) was incubated for the times indicated followed by Western blotting. (E) Striatal neurons were incubated with 10-NCP for 12 hours along with transduction of CellLight Mitochondria GFP to allow labeling and visualization of mitochondria. (F) Frequency distribution of mitochondrial size in control and 10-NCP treated neurons as quantitated from confocal immunofluorescent images using the Mitochondrial Morphology plug-in for ImageJ (n > 500 for each condition, X-axis truncated at 200). (G and H) Box and whisker plot (with whiskers representing 5-95th percentile) of the average mitochondrial size and major axis per neuron (n = 20). *P < 0.05.
Mentions: Imbalanced mitochondrial fusion and fission has been well characterized in neurodegeneration [26]. Increased Drp1 levels in AD [18] and HD patients [27], and cellular data from PD models all suggest that increased Drp1 is associated with pathogenesis. With this and evidence for autophagy-mediated turnover of Drp1 in mind, we studied the possible effect of inducing autophagy in a primary neurons. The compound 10-NCP has been found to potently induce autophagy in primary neurons [28]. Using cultured E18 rat-derived striatal neurons, 10-NCP is able to reduce Drp1 levels in a dose-dependent manner with a corresponding increase in LC3-II levels (Figure 3A). This effect is also time dependent and Drp1 is effectively decreased by 24 hours of 10-NCP treatment (Figure 3B). The previous study of 10-NCP in neurons also identified several FDA-approved structural analogs, which were able to analogously induce autophagy [28]. We were interested to see if these compounds would act similarly to 10-NCP in these neurons. Both compounds tested (chlorpromazine and trifluoperazine) were able to decrease Drp1 in a similar time dependent manner to 10-NCP (Figure 3C-D). To confirm the functional relevance of Drp1 downregulation, E18 rat-derived striatal neurons were treated with vehicle or 10-NCP along with transduction of a mitochondrially-targeted GFP (Figure 3E). It appears that 10-NCP leads to decreased fission as mitochondria become more interconnected after treatment (Figure 3E). To quantify this effect, mitochondrial morphology was analyzed using a well-characterized image analysis program (ImageJ plug-in) [22]. After 10-NCP treatments, mitochondrial size is increased in a large percentage of the neuronal mitochondria (Figure 3F). To better quantify this effect, the average and distribution of mitochondrial size and length (defined by the major mitochondrial axis) in control and 10-NCP treated neurons is shown (Figure 3G-H). These data suggest the induction of autophagy in neurons is able to decrease Drp1 and this approach may be useful to decrease the aberrant Drp1 expression observed in neurodegeneration.

Bottom Line: We are able to decrease Drp1 levels in a time- and dose-dependent manner with the potent neuronal autophagy inducer 10-NCP, as well as structurally related compounds.Further, 10-NCP was able increase average mitochondrial size and length verifying a functional result of Drp1 depletion in these neurons.Additionally these data suggest a mechanism, through Drp1 downregulation, which may partly explain the ability of autophagy to have a neuroprotective effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT

Background: Drp1 is the primary protein responsible for mitochondrial fission. Perturbations of mitochondrial morphology and increased fission are seen in neurodegeneration. While Drp1 degradation induced by Parkin overexpression can be prevented by proteasome inhibition, there are numerous links between proteasomal and autophagic processes in mitochondrial protein degradation. Here we investigated the role of autophagy in Drp1 regulation.

Results: We demonstrate that autophagy plays a major role in the control of Drp1 levels. In HEK-293T cells, inhibitors of autophagy increase total Drp1 and levels of Drp1 in the mitochondrial cellular fraction. Similarly by silencing ATG7, which is required for initiation of autophagy, there is an increased level of Drp1. Because of the role of increased Drp1 in neurodegeneration, we then examined the ability to modulate Drp1 levels in neurons by inducing autophagy. We are able to decrease Drp1 levels in a time- and dose-dependent manner with the potent neuronal autophagy inducer 10-NCP, as well as structurally related compounds. Further, 10-NCP was able increase average mitochondrial size and length verifying a functional result of Drp1 depletion in these neurons.

Conclusions: These pharmacological and genetic approaches indicate that autophagy targets Drp1 for lysosomal degradation. Additionally these data suggest a mechanism, through Drp1 downregulation, which may partly explain the ability of autophagy to have a neuroprotective effect.

Show MeSH
Related in: MedlinePlus