Role of 4-HNE metabolism in aging (Drosophila) (R01 grant) Role of glutathione transferases in life span extension of C. elegans Role of 4-HNE metabolism in aging (Drosophila and C. elegans) (Project 2 of P01) Role of glutathione transferases in life span extension of D. melanogaster

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal Ayyadevara, S., Engle, M.R., Singh, S.P., Dandapat, A., Lichti, C.F., Beneš, H., Shmookler Reis, R.J., Liebau, E. and Zimniak, P. (Aging Cell 4: , 2005) Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2 Ayyadevara, S., Dandapat, A., Singh, S.P., Beneš, H., Zimniak, L., Shmookler Reis, R.J. and Zimniak, P. (Aging Cell 4: , 2005) Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4­ hydroxynon-2-enal Ayyadevara, S., Dandapat, A., Singh, S.P., Siegel, E.R., Shmookler Reis, R.J., Zimniak, L. and Zimniak, P. (Mech. Ageing Dev., 2006, in press)

Hypothesis: adductscrosslinksmutations aging GST conjugate mGSTA4-4 hGSTA4-4 CeGSTP2-2 DmGSTS1-1 DmGSTD1-1

Reactive aldehydes can modify proteins

4-HNE conjugation (and the responsible GST) correlate with life span

To establish whether 4-HNE-conjugating activity and/or the responsible GSTs only CORRELATE with life span, or whether there is a CAUSE-EFFECT relationship, the levels of 4-HNE-conjugating GSTs were modulated by RNAi knockdown or by transgenic expression.

RNAi for CeGSTP2-2 (gst-10 gene product) is effective

RNAi against gst-10 but not against gst-8 shortens life span (gst-10 encodes CeGSTP2-2)

Expression of mGSTA4-4 or CeGSTP2-2 in transgenic worms

Overexpression of mGSTA4-4 and CeGSTP2-2 extends life span

Effect of mGSTA4-4 or CeGSTP2-2 on life span

Gompertz function

Compiled data for 4-HNE-conjugating activity, 4-HNE adducts, and life span (stress resistance shows similar behavior)

Effect of daf-2 on CeGSTP2-2 is dependent on daf-16

CeGSTP2-2 is localized to a limited set of cells (amphids/phasmids?)

Functions of amphids and phasmids David M. Tobin, Cornelia I. Bargmann, Invertebrate Nociception: Behaviors, Neurons and Molecules. J Neurobiol 61: 161–174, Alcedo, J. and Kenyon, C. (2004). Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron 41:

Possible mechanism by which CeGSTP2-2 extends life span

Do any of C. elegans GSTs other than CeGSTP2-2 affect aging?

5 GSTs have 4-HNE-conjugating activity (by RNAi screen) The above 5 GSTs may be responsible for most (or all) of 4-HNE conjugation

RNAi appears to be specific Based on sequence analysis (for gst-10) and Western blot (effect of a series of RNAi on gst-10)

4-HNE stress resistance: gst-6 and gst-24 are most effective

4-HNE-conjugating activity correlates well with 4-HNE stress resistance (10 mM 4-HNE)

C. elegans cuticle shields against 4-HNE. Therefore, 10 mM 4-HNE is NOT excessive - in fact, it is equivalent to 10 micromolar 4-HNE in cultured cells

4-HNE adducts change with age in C. elegans

RNAi targeted to gst-5 and gst-10 shortens life span RNAi targeted to gst-6, gst-8, and gst-24 does not shorten life span

Correlation between life span and 4-HNE- conjugating activity is poor: gst-6 and gst-24 gene products have the highest enzymatic activity gst-5 and gst-10 gene products exert the largest effect on life span Possible explanations: 1.4-HNE is not important 2.4-HNE AND localization are important

Expression of CeGSTP2-2 is limited to a small number of cells Where is gst-5 localized? gst-6 and gst-24?

Unrooted radial phylogenetic tree of C. elegans GSTs

D. melanogaster system:  Disruption of the GstS1 gene (encoding a GST responsible for most of 4-HNE-conjugating activity in the fly) leads to a moderate shortening of life span  Transgenic overexpression of the murine mGSTA4-4 (which has high catalytic efficiency for 4-HNE) in some but not all tissues of the fly leads to life span extension  Overall, the D. melanogaster and C. elegans systems confirm a role of 4-HNE in life span determination in two species that diverged approximately one billion years ago. The D. melanogster data are being prepared for publication