Mol. Cells 2017; 40(11): 864-870
Published online October 27, 2017
https://doi.org/10.14348/molcells.2017.0172
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: renocho@chosun.ac.kr
The uncoupling protein 4 (
Keywords aging,
Age-related diseases represent a major public health issue worldwide, as the overall incidence of such diseases has increased due to increases in the average lifespan (Prince et al., 2013). Among such diseases are neurodegenerative conditions such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease—all of which are associated with mitochondrial dysfunction (Bossy-Wetzel et al., 2008; Kerr et al., 2017; Villace et al., 2017). Moreover, recent research has revealed that accumulation of dysfunctional mitochondria in neurons is associated with neurodegeneration (Kerr et al., 2017). Increased levels of reactive oxygen species (ROS) and ATP deficits are prominent features of dysfunctional mitochondria in aged neurons (Grimm and Eckert, 2017). Since neuronal cells require high amounts of energy to perform their various functions, maintaining a healthy mitochondrial population is critical for cell survival (Rugarli and Langer, 2012).
In order to maintain healthy mitochondrial population, mitochondria contain several quality control mechanisms including mitophagy, an autophagy for mitochondrial removal (Ashrafi and Schwarz, 2013). Mitophagy is activated when mitochondrial damage cannot be repaired, or when the number of mitochondria in a cell surpasses the amount required (Ashrafi and Schwarz, 2013; Palikaras et al., 2015). Recent studies involving manipulation of uncoupling proteins in
Wild type of the Bristol N2 strain,
Individual neurons were categorized as defective when they exhibited a single structural abnormality, such as an outgrowth in anterior lateral microtubule (ALM) cells or wavy, neuronal sprout, and branching in posterior lateral microtubule (PLM) cells. All observations were performed using a fluorescent microscope (80i-DS-Fi1, Nikon). Unpaired
DNP and CCCP in absolute ethanol were added at the desired final concentration before pouring the plates: 100 μM DNP and 10 μM CCCP. 500 μM DNP and 50 μM CCCP were added after bacteria had been seeded onto the plates due to inhibition of bacterial growth. Worms in the plates were continuously exposed to DNP and CCCP throughout the lifetime.
Tetramethylrhodamine (TMRE, Molecular Probes) is a cell-permeable and cationic fluorescent dye that is commonly used as an indicator of mitochondrial membrane potential (Ψm) (Ehrenberg et al., 1988; Farkas et al., 1989). TMRE in DMSO (50 μM) was applied to worm plates at a final concentration of 0.1 μM. The plates were then incubated for 16 h, following which they were washed with M9 buffer and prepared for observation in accordance with previously described methods (Yoneda et al., 2004). For ectopic overexpression experiments, heat shock was performed for 2 h at 25°C, 12 h prior to observation.
In our previous study, we demonstrated that neuronal defects are associated with increased mitochondrial membrane potentials in
In the present study, mechanosensory touch receptor neurons, ALM and PLM cells, were labeled in the
If membrane uncoupling attenuates neurodegeneration, the effects of chemical uncouplers should be observed in animals with relevant genetic modifications. Thus, we examined the effects of such treatment in animals overexpressing
The
To determine whether phenotypic improvements observed in the present study resulted from reductions in mitochondrial membrane potentials, TMRE was used as an indicator of mitochondrial membrane potential (Yoneda et al., 2004). As shown in Fig. 3, animals treated with DNP (100 μM and 500 μM) or CCCP (10 μM and 50 μM) exhibited decreased TMRE intensity relative to that observed in wild type controls on day 1 (Figs. 3A–3E). A positive association was observed between TMRE intensity and chemical concentration in uncoupler-treated animals: TMRE intensity in 500 μM DNP or 50 μM CCCP-treated animals was lower than the intensity in 100 μM DNP or 10 μM CCCP-treated animals (Figs. 3B–3E). Substantially lower TMRE staining was observed in
TMRE staining patterns in aged animals were similar to those observed on day 1, lower intensity in
Recent research has indicated that depolarization of the mitochondrial membrane potential activates mitophagy, especially via the PINK1/Parkin pathway (MacVicar and Lane, 2014; Narendra et al., 2008; 2010). Furthermore, age-related impairments in mitophagy have been associated with neurodegenerative symptoms in patients with Alzheimer’s, Parkinson’s, and Huntington’s disease (Itoh et al., 2013). Therefore, we investigated whether mitophagy plays a role in age-dependent neurodegeneration via reductions in mitochondrial membrane potential. Experiments in mitophagy-deficient mutants,
Progressive increases in the number of ALM defects were observed in
In summary, the findings of the present study indicate that mitochondrial uncoupling attenuated neuronal defects during aging in
Mol. Cells 2017; 40(11): 864-870
Published online November 30, 2017 https://doi.org/10.14348/molcells.2017.0172
Copyright © The Korean Society for Molecular and Cellular Biology.
Injeong Cho1,3, Hyun-Ok Song2,3, and Jeong Hoon Cho1,*
1Department of Biology Education, College of Education, Chosun University, Gwangju 61452, Korea, 2Department of Infection Biology, Wonkwang University School of Medicine, Iksan 54538, Korea
Correspondence to:*Correspondence: renocho@chosun.ac.kr
The uncoupling protein 4 (
Keywords: aging,
Age-related diseases represent a major public health issue worldwide, as the overall incidence of such diseases has increased due to increases in the average lifespan (Prince et al., 2013). Among such diseases are neurodegenerative conditions such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease—all of which are associated with mitochondrial dysfunction (Bossy-Wetzel et al., 2008; Kerr et al., 2017; Villace et al., 2017). Moreover, recent research has revealed that accumulation of dysfunctional mitochondria in neurons is associated with neurodegeneration (Kerr et al., 2017). Increased levels of reactive oxygen species (ROS) and ATP deficits are prominent features of dysfunctional mitochondria in aged neurons (Grimm and Eckert, 2017). Since neuronal cells require high amounts of energy to perform their various functions, maintaining a healthy mitochondrial population is critical for cell survival (Rugarli and Langer, 2012).
In order to maintain healthy mitochondrial population, mitochondria contain several quality control mechanisms including mitophagy, an autophagy for mitochondrial removal (Ashrafi and Schwarz, 2013). Mitophagy is activated when mitochondrial damage cannot be repaired, or when the number of mitochondria in a cell surpasses the amount required (Ashrafi and Schwarz, 2013; Palikaras et al., 2015). Recent studies involving manipulation of uncoupling proteins in
Wild type of the Bristol N2 strain,
Individual neurons were categorized as defective when they exhibited a single structural abnormality, such as an outgrowth in anterior lateral microtubule (ALM) cells or wavy, neuronal sprout, and branching in posterior lateral microtubule (PLM) cells. All observations were performed using a fluorescent microscope (80i-DS-Fi1, Nikon). Unpaired
DNP and CCCP in absolute ethanol were added at the desired final concentration before pouring the plates: 100 μM DNP and 10 μM CCCP. 500 μM DNP and 50 μM CCCP were added after bacteria had been seeded onto the plates due to inhibition of bacterial growth. Worms in the plates were continuously exposed to DNP and CCCP throughout the lifetime.
Tetramethylrhodamine (TMRE, Molecular Probes) is a cell-permeable and cationic fluorescent dye that is commonly used as an indicator of mitochondrial membrane potential (Ψm) (Ehrenberg et al., 1988; Farkas et al., 1989). TMRE in DMSO (50 μM) was applied to worm plates at a final concentration of 0.1 μM. The plates were then incubated for 16 h, following which they were washed with M9 buffer and prepared for observation in accordance with previously described methods (Yoneda et al., 2004). For ectopic overexpression experiments, heat shock was performed for 2 h at 25°C, 12 h prior to observation.
In our previous study, we demonstrated that neuronal defects are associated with increased mitochondrial membrane potentials in
In the present study, mechanosensory touch receptor neurons, ALM and PLM cells, were labeled in the
If membrane uncoupling attenuates neurodegeneration, the effects of chemical uncouplers should be observed in animals with relevant genetic modifications. Thus, we examined the effects of such treatment in animals overexpressing
The
To determine whether phenotypic improvements observed in the present study resulted from reductions in mitochondrial membrane potentials, TMRE was used as an indicator of mitochondrial membrane potential (Yoneda et al., 2004). As shown in Fig. 3, animals treated with DNP (100 μM and 500 μM) or CCCP (10 μM and 50 μM) exhibited decreased TMRE intensity relative to that observed in wild type controls on day 1 (Figs. 3A–3E). A positive association was observed between TMRE intensity and chemical concentration in uncoupler-treated animals: TMRE intensity in 500 μM DNP or 50 μM CCCP-treated animals was lower than the intensity in 100 μM DNP or 10 μM CCCP-treated animals (Figs. 3B–3E). Substantially lower TMRE staining was observed in
TMRE staining patterns in aged animals were similar to those observed on day 1, lower intensity in
Recent research has indicated that depolarization of the mitochondrial membrane potential activates mitophagy, especially via the PINK1/Parkin pathway (MacVicar and Lane, 2014; Narendra et al., 2008; 2010). Furthermore, age-related impairments in mitophagy have been associated with neurodegenerative symptoms in patients with Alzheimer’s, Parkinson’s, and Huntington’s disease (Itoh et al., 2013). Therefore, we investigated whether mitophagy plays a role in age-dependent neurodegeneration via reductions in mitochondrial membrane potential. Experiments in mitophagy-deficient mutants,
Progressive increases in the number of ALM defects were observed in
In summary, the findings of the present study indicate that mitochondrial uncoupling attenuated neuronal defects during aging in
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