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- Materials and methods
Damage from reactive oxygen species (ROS) is thought to be a cause of organismal aging. Reactive oxygen species have also been proposed to be responsible for several age-associated phenotypes, including age-related memory impairment (AMI). However, it has not previously been tested whether increasing ROS affects AMI onset. Here we examined the effects of feeding hydrogen peroxide, and the ROS-generating agent, paraquat, on olfactory aversive memory in Drosophila at young ages and during AMI onset. Reactive oxygen species feeding greatly reduced fly survival, and increased oxidized proteins and transcripts of an antioxidant enzyme, catalase (Cat) and a stress-responsive chaperone, heat-shock protein 22 (Hsp22) in fly heads. However, feeding did not impair memory in young wild-type flies, nor did it exacerbate the memory deficits in flies at the onset of AMI. Strikingly ROS feeding did disrupt memory at young ages and accelerated AMI onset was observed when expression of genes involved in the defense system to ROS, including antioxidant enzymes and Hsp22, was reduced in the mushroom bodies, neural centers required for olfactory memory. These results implicate that although ROS production increases upon aging, neuronal functions required for memory processes are sufficiently protected by the defense system to ROS even at the age of AMI onset. Thus we propose that ROS production does not affect AMI onset in Drosophila.
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide (H2O2) and hydroxyl radicals are produced during cellular energy production. Reactive oxygen species damage cellular macromolecules such as nucleic acids, lipids and proteins, thereby disrupting cellular functions. Since generation of ROS is thought to increase upon aging, a free radical hypothesis has been proposed that cumulative oxidative damage produced by ROS causes aging processes and limits life span (Balaban et al. 2005; Beckman & Ames 1998; Harman 1956). However, due to a defense system against ROS (anti-ROS system), ROS are normally scavenged by antioxidant enzymes such as superoxide dismutase (Sod), catalase (Cat) and glutathione peroxidase (Balaban et al. 2005), and damaged proteins are thought to be repaired by stress-responsive molecular chaperones such as heat-shock proteins (Hsps) (Parsell & Lindquist 1993). Thus, the free radical hypothesis predicts that the normal anti-ROS system is not fully sufficient to counteract the deleterious effects caused by ROS in aged organisms. In support of this idea, overexpressing sod1 and sod2 (Parkes et al. 1998; Sun et al. 2002) and feeding Sod/Cat mimetics (Melov et al. 2000) extend the life span of Drosophila and Caenorhabditis elegans. In Drosophila, lack of hsp22 shortens life span (Morrow et al. 2004a), while overexpression of hsp22 extends life span and increases resistance to oxidative damage (Morrow et al. 2004b).
Although it has not been proven that factors affecting life span also affect age-related memory impairment (AMI) (Burger et al. 2010; Horiuchi & Saitoe 2005), prevailing models propose that AMI is also caused by ROS (Bishop et al. 2010). In mammals, age-related accumulation of oxidative damage is observed in the brain (Lu et al. 2004; Murali & Panneerselvam 2007b; Smith et al. 1991), and genetic and pharmacological interventions that enhance activity of the anti-ROS system improve memory of old animals (Hu et al. 2006; Levin et al. 2005; Liu et al. 2003). AMI is also observed in Drosophila, which has a life span of 30–40 days. Using a Pavlovian olfactory aversive association task (Tully & Quinn 1985), we previously showed that AMI in Drosophila appears at 15 days after eclosion, and consists of a significant and specific reduction in middle-term memory (MTM) which can be measured as memory 1 h after single-cycle training (Tamura et al. 2003). However, it is not clear whether ROS are involved in induction of AMI in Drosophila and to what extent the anti-ROS system counteracts the deleterious effects of ROS on memory.
To address these issues, we fed flies with ROS at young ages and at the age of AMI onset. Our results show that neuronal functions required for memory formation are highly protected from ROS by the anti-ROS system. We provide data implicating that although amounts of ROS increase upon aging, ROS seem not contribute to AMI onset due to a robust anti-ROS system.
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- Materials and methods
In Drosophila, AMI is characterized by a specific reduction in MTM, a memory phase that can be measured 1 h after single-cycle training. Age-related memory impairment onset occurs at 15 days of age and shows an increase in severity upon further aging. Although cumulative damage caused by ROS has been implicated in AMI (Levin et al. 2005; Liu et al. 2003; Murali & Panneerselvam 2007a; Nicolle et al. 2001), we provide data suggesting that AMI onset is not induced by ROS in Drosophila. If ROS cause AMI, an increase in ROS should further enhance AMI. However, neither acute nor chronic feeding with PQ and H2O2 enhanced AMI at 15 days of age, although they caused oxidative damage, decreased life span and induced anti-ROS responses.
Our present data implicate that neuronal functions required for MTM formation are highly protected from ROS due to a robust anti-ROS system which includes Cat, Sod1, Sod2 and Hsp22 by the age of AMI onset. While PQ or H2O2 feeding did not affect memory in wild-type flies, RNAi lines with reduced hsp22, cat and sod1 expression had memory defects after PQ and H2O2 feeding at 3 days of age. They also showed age-dependent memory defects at 10 days of age in the absence of PQ and H2O2 feeding, an age prior to wild-type AMI onset. Since expression of hsp22 and cat increase after feeding PQ and H2O2, activity of the anti-ROS system is likely to be upregulated upon increasing ROS even at 15 days of age. Notably, in contrast to 3-day-old RNAi transgenic flies, 10-day-old RNAi transgenic flies displayed memory defects in the absence of PQ and H2O2 feeding. Taken together, these results suggest that the amount of ROS increases in fly heads upon aging. However, this increase fails to induce memory defects at the age of AMI onset due to increased activity of the anti-ROS system.
Our data show that ROS cause memory defects when activity of the anti-ROS system is decreased. In line with this, observed AMI in rodents is accompanied by decreases in the anti-ROS system, including decreases in glutathione concentration (Murali & Panneerselvam 2007a; Zhu et al. 2006) and decreases in activity of Cat (Tian et al. 1998) and Sod (Gupta et al. 1991). In addition, mammalian AMI is ameliorated by enhancing the anti-ROS system by methods including sod overexpression (Hu et al. 2006; Levin et al. 2005) and administration of Sod/Cat mimetics (Liu et al. 2003). In our hands, overexpression of sod1 did not suppress AMI onset in Drosophila (data not shown) indicating that while an age-related increase in ROS is likely to contribute to mammalian AMI, it may not do so to AMI onset in flies. As AMI onset is significantly delayed by reducing Protein kinase A (PKA) activity in the MBs (Yamazaki et al. 2007; Yamazaki et al. 2010), our current results implicate that age-related increase in PKA-dependent signaling plays critical role in the initiation of AMI in a ROS-independent manner.
It is still not clear how ROS disrupt memory formation. Oxidative damage is often linked to neurodegeneration (Floyd 1999). However, feeding PQ did not induce neuronal apoptosis in the brain of RNAi transgenic flies (Hirano unpublished observation). Therefore, neurodegeneration cannot account for memory defects observed in RNAi transgenic flies. Intriguingly Hsp22 is localized in mitochondria (Morrow et al. 2000). As mitochondria play important roles in memory formation (Ben-Shachar & Laifenfeld 2004), Hsp22 may repair damaged mitochondrial proteins required for memory formation.
While our current study suggests that ROS are not involved in the initial onset of AMI, we do not exclude the possibility that ROS are involved in later aspects of AMI including regulation of AMI severity. Notably, although expression of hsp22 increases at the age of AMI onset, expression decreases in fly heads upon further aging (Landis et al. 2004). This suggests that the anti-ROS system might eventually be overwhelmed by ROS leading to increased severity of AMI.