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Overexpression of the small mitochondrial Hsp22 extends Drosophila life span and increases resistance to oxidative stress¹

^* Laboratory of Cell and Developmental Genetics, CREFSIP and Department Medicine, Université Laval, Ste-Foy, Québec, Canada

³Correspondence: Laboratory of Cell and Developmental Genetics, Pavillon Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4. E-Mail: robert.tanguay{at}rsvs.ulaval.ca

SPECIFIC AIM

Small heat shock proteins (sHsp) are molecular chaperones that can prevent protein aggregation and assist protein refolding in vitro. To determine the in vivo role of the small mitochondrial heat shock protein Hsp22, we examined the effects of overexpressing this sHsp on aging and resistance to stress in Drosophila melanogaster.

PRINCIPAL FINDINGS

1. Expression of Hsp22 increases mean life span
The GAL4/UAS system was used to drive the expression of the endogenous hsp22 gene in distinct cells. Flies overexpressing Hsp22 ubiquitously (actin driver) or in motorneurons (D42 driver) displayed a 30% increase in mean life span (Fig. 1 ). The shapes of the longevity curves suggest a role of Hsp22 in early-aging events since the increase in the length of the premortality phase is the same as that of the mean life span. Once the mortality phase is reached, overexpression of Hsp22 does not seem to have any beneficial effect as seen by the similar slope of the survival curves of Hsp22 expressing and control flies. These data demonstrate the protective effect of Hsp22 against aging.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of targeted expression of Hsp22 on life span. Longevity curves of adult males expressing Hsp22 (filled squares) (A) ubiquitously (EP(3)3247/actin-GAL4) or B) in motorneurons (EP(3)3247/D42-GAL4) and of their matched controls (+/actin-GAL4; +/D42-GAL4) (open triangles).

2. Flies expressing Hsp22 in their motorneurons maintain their locomotor activity longer
To determine whether the extension of life span was associated with change in locomotor activity, we used a negative geotaxis assay at 40, 60, and 80 days of age. Targeting expression of Hsp22 in motorneurons allows flies to maintain a normal locomotor activity longer. At day 60, control flies (+/D42-GAL4) entering the mortality phase show a decreased locomotor activity of 10% while flies expressing Hsp22 in motorneurons (EP(3)3247/D42-GAL4) retain a normal activity (28%). Flies expressing Hsp22 under the control of the ubiquitous actin promoter loose activity in the premortality phase but still maintain 30% activity until day 80. These data indicate that flies expressing Hsp22 retain locomotor activity longer and suggest that the integrity of motorneurons is a key factor for maintenance of this activity.

3. Targeted expression of Hsp22 in motorneurons protects against oxidative and thermal stress
Since aging has been associated with accumulation of oxidative damages, we tested whether expression of Hsp22 conferred resistance to an oxidative stress induced by paraquat. Flies overexpressing Hsp22 in their motorneurons were more resistant than controls flies at all ages tested (Fig. 2 ). The largest increase in resistance was observed on day 2 when flies expressing Hsp22 were 35% more resistant. Targeted overexpression of Hsp22 in motorneurons was also responsible for a 39% increase in mean life span after incubation at 30°C and a 23% increase when flies were kept at 37°C. These data demonstrate that expression of the small mitochondrial Hsp22 in motorneurons is sufficient to increase resistance to thermal and oxidative stresses.

View larger version (17K): [in this window] [in a new window]   Figure 2. Resistance to the ROS-generating paraquat increases in flies expressing Hsp22 in motorneurons. Survival of EP(3)3247/D42-GAL4 (black) and control +/D42-GAL4 flies (white) of different ages 24 h postparaquat treatment. 2- and 40-day-old flies received 20 mM paraquat whereas 60- and 80-day-old flies were submitted to 1 mM paraquat.

CONCLUSIONS AND SIGNIFICANCE

The present study shows that overexpression of the mitochondrial chaperone Hsp22 can increase both life span and resistance to oxidative and thermal stress. The increase varies depending on the cells targeted for overexpression. Despite being highly localized to a restricted number of cells, motorneuron-targeted expression of Hsp22 increased longevity by 32% and survival by 35% in a paraquat resistance assay. These results not only confirm the key role of motorneurons in the aging process but also support their sensitivity tooxidative stress. Overexpression of transgenic human Cu/Zn-SOD in the same cells has been reported to increase life span and resistance to oxidative stress.

The effectiveness of mitochondrial Hsp22 to increase life span may reflect the particular sensitivity of mitochondria and the importance of maintaining their integrity in the aging process. Overexpressing the cytoplasmic chaperones Hsp23 or Hsp26 resulted in a maximal 15% increase in mean life span. The beneficial effect of Hsp22 could also explain why its expression is constitutively and preferentially up-regulated in aging flies.

The observation that the increase in longevity is observed mainly during the premortality phase suggests that Hsp22 plays a role in early aging events. Since aging is associated with increased protein oxidation and modification, Hsp22 could act by chaperoning damaged proteins, hence preventing their accumulation in toxic insoluble aggregates and their subsequent deleterious effect on mitochondrial integrity and function (Fig. 3 ). In vitro Hsp22 is an efficient chaperone, but its function in vivo is unknown.

View larger version (36K): [in this window] [in a new window]   Figure 3. Potential targets of Hsp22 action in mitochondria during aging. Post-transcriptional modifications of proteins by ROS increase the pool of aggregated and/or inactive proteins. Hsp22 could be constitutively up-regulated in aging flies to prevent aggregation of non-native proteins through its chaperones properties.

In summary, the present study provides a link between expression of a mitochondrial chaperone, resistance to oxidative stress, and longevity and is consistent with a model in which the relationship between thermal stress and life span is based upon the rate at which altered proteins accumulate during thermal stress and during aging. The data also establish Hsp22, a mitochondrial chaperone capable of preventing heat-induced denaturation and maintaining misfolded proteins in a refolding competent state, as an important player in cell protection mechanisms against oxidative and environmental injuries and aging.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0860fje

² Present address: Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115, USA.

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