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Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons.

Cavallucci V, Bisicchia E, Cencioni MT, Ferri A, Latini L, Nobili A, Biamonte F, Nazio F, Fanelli F, Moreno S, Molinari M, Viscomi MT, D'Amelio M - Cell Death Dis (2014)

Bottom Line: A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability.In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion.Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Neurosciences, IRCCS S. Lucia Foundation, Rome, Italy.

ABSTRACT
Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.

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Rapamycin treatment improves mitochondrial function and perturbs mitochondria dynamics. (a) Representative cytofluorimetric analysis (left) and quantification graphs (right) of mitochondria isolated from precerebellar nuclei of saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice at 24 h and stained with tetramethylrhodamine ethyl ester (TMRE). Data are expressed as mean ±S.D. (**P<0.01, n=4). (b) Representative immunoblots and densitometric graph of mitochondrial levels of Drp, Mfn1, PINK1, Parkin, and p62 in precerebellar nuclei of saline- and rapamycin-treated Ctrl (Ctrl+S and Ctrl+R24h, respectively) and saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice. The mitochondrial protein Tom20 was used as loading control. Data are expressed as mean±S.D. (**P<0.01, ***P<0.001, n=7). Ctrl+S is indicated as 100%
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fig4: Rapamycin treatment improves mitochondrial function and perturbs mitochondria dynamics. (a) Representative cytofluorimetric analysis (left) and quantification graphs (right) of mitochondria isolated from precerebellar nuclei of saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice at 24 h and stained with tetramethylrhodamine ethyl ester (TMRE). Data are expressed as mean ±S.D. (**P<0.01, n=4). (b) Representative immunoblots and densitometric graph of mitochondrial levels of Drp, Mfn1, PINK1, Parkin, and p62 in precerebellar nuclei of saline- and rapamycin-treated Ctrl (Ctrl+S and Ctrl+R24h, respectively) and saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice. The mitochondrial protein Tom20 was used as loading control. Data are expressed as mean±S.D. (**P<0.01, ***P<0.001, n=7). Ctrl+S is indicated as 100%

Mentions: Thus, to evaluate the effect of autophagy stimulation on the removal of damaged mitochondria 24 h after HCb, we isolated mitochondria of axotomized precerebellar nuclei from mice that were treated with a single dose of rapamycin (HCb24h+R) or saline solution (HCb24h+S) as Ctrl (Table 1). Mitochondrial TMRE staining revealed that the fraction of polarized mitochondria from rapamycin-treated mice exceeded that of mice that were injected with saline (P<0.01, HCb24h+S versus HCb24h+R; Figure 4a). As Ctrl, all mitochondrial preparations were also stained with TMRE in the presence of FCCP (Supplementary Figure 1d). This result suggests that the protective effect of rapamycin is attributed to its ability to stimulate the removal of damaged mitochondria. We have demonstrated26 a lack of efficacy of rapamycin in mice with impaired autophagic responses (Beclin heterozygous mice), implicating autophagy mechanisms as the principal target of rapamycin-associated neuroprotective effects.


Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons.

Cavallucci V, Bisicchia E, Cencioni MT, Ferri A, Latini L, Nobili A, Biamonte F, Nazio F, Fanelli F, Moreno S, Molinari M, Viscomi MT, D'Amelio M - Cell Death Dis (2014)

Rapamycin treatment improves mitochondrial function and perturbs mitochondria dynamics. (a) Representative cytofluorimetric analysis (left) and quantification graphs (right) of mitochondria isolated from precerebellar nuclei of saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice at 24 h and stained with tetramethylrhodamine ethyl ester (TMRE). Data are expressed as mean ±S.D. (**P<0.01, n=4). (b) Representative immunoblots and densitometric graph of mitochondrial levels of Drp, Mfn1, PINK1, Parkin, and p62 in precerebellar nuclei of saline- and rapamycin-treated Ctrl (Ctrl+S and Ctrl+R24h, respectively) and saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice. The mitochondrial protein Tom20 was used as loading control. Data are expressed as mean±S.D. (**P<0.01, ***P<0.001, n=7). Ctrl+S is indicated as 100%
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260762&req=5

fig4: Rapamycin treatment improves mitochondrial function and perturbs mitochondria dynamics. (a) Representative cytofluorimetric analysis (left) and quantification graphs (right) of mitochondria isolated from precerebellar nuclei of saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice at 24 h and stained with tetramethylrhodamine ethyl ester (TMRE). Data are expressed as mean ±S.D. (**P<0.01, n=4). (b) Representative immunoblots and densitometric graph of mitochondrial levels of Drp, Mfn1, PINK1, Parkin, and p62 in precerebellar nuclei of saline- and rapamycin-treated Ctrl (Ctrl+S and Ctrl+R24h, respectively) and saline- and rapamycin-treated HCb (HCb24h+S and HCb24h+R, respectively) mice. The mitochondrial protein Tom20 was used as loading control. Data are expressed as mean±S.D. (**P<0.01, ***P<0.001, n=7). Ctrl+S is indicated as 100%
Mentions: Thus, to evaluate the effect of autophagy stimulation on the removal of damaged mitochondria 24 h after HCb, we isolated mitochondria of axotomized precerebellar nuclei from mice that were treated with a single dose of rapamycin (HCb24h+R) or saline solution (HCb24h+S) as Ctrl (Table 1). Mitochondrial TMRE staining revealed that the fraction of polarized mitochondria from rapamycin-treated mice exceeded that of mice that were injected with saline (P<0.01, HCb24h+S versus HCb24h+R; Figure 4a). As Ctrl, all mitochondrial preparations were also stained with TMRE in the presence of FCCP (Supplementary Figure 1d). This result suggests that the protective effect of rapamycin is attributed to its ability to stimulate the removal of damaged mitochondria. We have demonstrated26 a lack of efficacy of rapamycin in mice with impaired autophagic responses (Beclin heterozygous mice), implicating autophagy mechanisms as the principal target of rapamycin-associated neuroprotective effects.

Bottom Line: A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability.In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion.Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Neurosciences, IRCCS S. Lucia Foundation, Rome, Italy.

ABSTRACT
Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.

Show MeSH
Related in: MedlinePlus