PerspectiveCell Signaling

Mitochondrial Longevity Pathways

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Science  02 Feb 2007:
Vol. 315, Issue 5812, pp. 607-609
DOI: 10.1126/science.1138825

The quest for longevity has led to the discovery of several genes that affect the life span of organisms ranging from yeast to mammals. An increased life span has been linked to the expression of sirtuins, impaired function insulin receptor homologs, and absence of the signaling protein p66Shc. Several cell signaling pathways associated with these factors converge on the Forkhead/FOXO family of transcription factors, which regulate the expression of a battery of stress response proteins that affect antioxidant capacity, cell cycle arrest, DNA repair, and apoptosis (1). Life span-regulating proteins also directly affect mitochondrial function, including energy metabolism and reactive oxygen species production, in which p66Shc plays a critical role. How these mitochondrial processes integrate with the upstream signaling events to control life span has remained enigmatic. On page 659 of this issue, Pinton et al. describe a signaling pathway that controls the mitochondrial activity of p66Shc (2) (see the figure) and provides insight into how this integration might occur.

The extended life span of mice lacking p66Shc has been correlated with a decrease in mitochondrial metabolism (3) and reactive oxygen species production (4). Pinton et al. show that p66Shc is required for early mitochondrial responses to an oxidative challenge (hydrogen peroxide, H2O2). These responses include mitochondrial fragmentation and suppression of Ca2+ signal propagation to the mitochondria, followed by execution of apoptosis (cell death) in murine fibroblasts. The authors found that early mitochondrial response to H2O2 increased progressively with cell culture age, and used this model to map the signaling cascade through which p66Shc affects mitochondria.

Reactive oxygen species affect the activity of many protein phosphatases and kinases. Phosphorylation of p66Shc on Ser36 can be mediated by several protein kinases and is indispensable for life-span regulation by p66Shc. Pinton et al. show that inhibiting or silencing protein kinase C β protects cells against H2O2 challenge. Furthermore, overexpression of protein kinase C β reproduces the mitochondrial fragmentation and Ca2+ signaling defect in cells expressing p66Shc, but not in cells lacking p66Shc. Cells expressing a mutant form of p66Shc (p66ShcS36A) that cannot be phosphorylated also lack the early mitochondrial response to protein kinase C β activity, indicating a requirement for p66Shc phosphorylation. Thus, there is remarkable interdependence between protein kinase C β and p66Shc in the mitochondrial response to oxidative challenges. Although H2O2 challenge seems to employ only protein kinase C β to phosphorylate p66Shc, this step may serve as an integration site for multiple protein kinases activated by cell surface receptors.

P66Shc localizes predominantly in the cytoplasm, with a smaller fraction (10 to 40%) in the mitochondrial intermembrane space (3, 5). The protein lacks a conventional mitochondrial targeting sequence, but its association with the mitochondrial TOM/TIM import complex and the mitochondrial heat shock protein mtHsp70 has been reported (5). Pinton et al. put forward the original idea that phosphorylation of Ser36 induces translocation of phosphorylated p66Shc to the mitochondria. They also provide evidence that both H2O2 challenge and protein kinase C β activation promote binding of p66Shc to Pin1, causing this translocation. Pin1 is a peptidylprolyl isomerase that induces cis-trans isomerization of phosphorylated Ser-Pro bonds. This confers phosphorylation-dependent conformational changes in Pin1 targets. Pin1 has been studied in the context of the processing of phosphorylated proteins in Alzheimer's disease (6), but the present data suggest that it has broader importance. Binding of phosphorylated p66Shc to Pin1 may expose a hidden sequence that targets p66Shc to the mitochondria. It is possible that this unconventional targeting mechanism might enable p66Shc to interact with a specific subset of a heterogeneous mitochondrial population, and provide a means for differential regulation of mitochondria by p66Shc.

Signal integration.

Phosphorylated p66Shc may serve as an integration point for many signaling pathways that affect mitochondrial function and longevity. The pathway described by Pinton et al. is marked in red. Protein kinase Cβ (PKCβ).

Activation of mitochondrial p66Shc requires its dephosphorylation and dissociation from mtHsp70, but it remains unclear as to whether dephosphorylation by the phosphatase PP2A occurs before, upon, or after translocation into the organelle. Once in the intermembrane space, p66Shc interacts with reduced cytochrome c to produce H2O2, which can promote opening of the mitochondrial permeability transition pore. The sensitivity of the suppression of mitochondrial Ca2+ signaling and fragmentation to inhibitors of the permeability transition pore shows that these effects of p66Shc are downstream of the pore opening. Interestingly, H2O2 formation in the mitochondria is required for pore opening even when cells are exposed to an H2O2 challenge. Perhaps p66Shc-mediated reactive oxygen species generation requires a favorable environment in the intermembrane space to convert H2O2 to more damaging hydroxyl radicals and/or provide access to hidden sulfhydryl groups that regulate the permeability transition pore (7).

How is permeabilization of the mitochondrial inner membrane associated with onset of the aging phenotype? In the model of Pinton et al., p66Shc supports an apoptotic response to a massive oxidative challenge, a classical consequence of permeability transition pore opening and the ensuing release of cytochrome c. Apoptosis and removal of seriously damaged cells may play a role in both extending and shortening the cell's life span. Apoptosis involves the entire mitochondrial population displaying a coordinated response throughout the cell (8). However, pore activation confined to a subpopulation of mitochondria could help remove impaired mitochondria by triggering their disposal by autophagy and degradation in lysosomes (9). Indeed, aging may be associated with impaired autophagy (10) and inhibiting autophagy prevents life-span extension in the nematode Caenorhabditis elegans (11). Thus, p66Shc-dependent mitochondrial fragmentation and suppression of Ca2+ uptake may be relevant to localize and attenuate mitochondria damage where reactive oxygen species are produced as part of a mitochondrial quality control mechanism. Mice lacking p66Shc apparently remain healthy in the laboratory setting (12), but their sensitivity to environmental stress remains to be established. Thus, p66Shc may protect the organism against stress, but if targeted destruction of mitochondria by p66Shc overwhelms the autophagy capacity of the cell, this would set the stage for the accumulation of unprocessed oxidative-damaged cell constituents, a classical correlate of aging.

This conclusion suggests that regulating autophagy of damaged mitochondria may constitute another piece of the aging puzzle. The serine-threonine kinase mTOR is a critical inhibitor of autophagy (10) and also enhances mitochondrial metabolism and reactive oxygen species generation (3). Therefore, p66Shc and mTOR may interact to integrate multiple aspects of cell homeostasis that are relevant for aging. Sirtuins also may affect mitochondrial function beyond the expression of FOXO-regulated antioxidant and proapoptotic proteins. Notably, PGC-1α, a transcriptional coactivator that controls mitochondrial biogenesis and energy metabolism, is regulated by the sirtuin SIRT1. Also, several sirtuins localize to mitochondria where they affect critical metabolic functions (1315). SIRT1 and other life span-controlling proteins also have direct links to the mitochondrial permeabilization and autophagy, perhaps through Bcl-2 family proteins (14).

The factors that set the stage for the mitochondrial contribution to the aging process include inputs from different directions. Reactive oxygen species production in mitochondria is regulated by metabolic activity, substrate supply, and mitochondrial membrane potential. Superimposed on this background, mitochondrial import of p66Shc can provide an additional reactive oxygen species-producing element to trigger a local permeability transition pore opening that (together with other inputs) controls life span. Of course, this does not exclude a role for mitochondrial p66Shc in normal cell management of stress and damage repair. In fact, a recent study showed that p66Shc is highly expressed in fibroblasts from centenarians (16). P66Shc may well keep us fit while helping us age.


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