Augmented autophagy pathways and MTOR modulation in fibroblasts from long-lived mutant mice

Abstract

Fibroblasts from long-lived pituitary dwarf mutants, including Snell dwarf, Ames dwarf and the growth hormone receptor knockout (GHRKO) mice, are resistant in culture to multiple forms of lethal stress. We found that fibroblasts from Snell dwarf and GHRKO mice are more susceptible than control cells to autophagy induced by amino acid withdrawal or by oxidative stress. We also found evidence for lower MTOR function in dwarf cells under conditions that induce autophagy, consistent with the evidence that increased autophagy requires lower TOR activity. Our results provide new hints about the connections between autophagy and aging in long-lived mutants with alterations in GH-IGF1 levels, and suggest a role for hyperactive autophagy in the resistance of cells from these mice to lethal stresses.

Mutations that interfere with production of, or response to, growth hormone and its mediator IGF1 often lead to slower aging and extended longevity in mice, consistent with results on the insulin/IGF1 signaling (“IIS”) pathway in worms, flies, and dogs. Alterations of IIS genes in invertebrates typically modulate a series of intracellular pathways involving FOXO, MTOR, NFKB/NFκB, sirtuins, and TP53/p53, each of which has, in turn, been shown to play a role in autophagy. In some cases, invertebrate mutants have been shown to require autophagy to produce a longevity benefit. The extent to which modulation of autophagy may play a critical role in the longevity effect in long-lived mouse mutants with reduction of GH-IGF1 pathways is still uncertain

Surprisingly, skin-derived fibroblast cells from at least three varieties of long-lived mice—the Snell dwarf, Ames dwarf, and GH receptor knockout mice—are resistant, in culture, to lethal injury caused by exposure to oxidative stresses like hydrogen peroxide and paraquat. This resistance presumably represents a stable property reflecting epigenetic changes secondary to differentiation in the low-hormone environment of the juvenile mouse and retained during explantation and serial culture. Reversal of the longevity phenotype of Ames dwarf mice by early life injection of GH also reverses the stress resistance of skin-derived fibroblasts from these mice. The ability to study stress resistance in cultured cells gave us an opportunity to compare Snell dwarf-derived to control fibroblasts under conditions that induce autophagy, such as amino acid withdrawal and exposure to hydrogen peroxide and paraquat. We found that cells from dwarf mice were more susceptible to autophagy induction than control cells induced by amino acid deprivation. Similar results are seen using fibroblasts from GHRKO mice, suggesting that alterations in GH and/or IGF1 signals, in the developing young mouse, lead to the alteration in cell behavior seen in the cultured cells.

We suspected that hypersensitivity to autophagy induction might reflect underlying alterations in the mechanistic target of rapamycin (MTOR) kinase, which provides negative regulation of autophagy. We therefore evaluated phosphorylation of MTOR and phosphorylation of its downstream substrates RPS6KB2/p70S6 protein kinase and the eukaryotic initiation factor 4E-binding protein 1 (EIF4EBP1/4EBP1). Consistent with our observations of upregulated autophagy, we noted a more dramatic decline in MTOR signaling in cells from Snell dwarf mice in response to amino acid withdrawal. A similar pattern was noted for cells from long-lived GHRKO mice. Adding bafilomycin A₁ to inhibit processing of autophagic vesicles increases the susceptibility of TOR signals to amino acid withdrawal, but does not eliminate the difference between dwarf and control cells.

We next sought to determine if differences in autophagy induction might play a role in the relative resistance of cells from long-lived dwarf mutants to oxidative stress. Both paraquat and hydrogen peroxide induce higher levels of autophagy in dwarf-derived cells than in control cells, a difference that was particularly marked in the presence of bafilomycin. Peroxide or paraquat upregulate MTOR signals only in control cells, but not in Snell dwarf cells, consistent with the idea that the stable, lower levels of MTOR in the dwarf cells might be responsible for their elevated autophagy after oxidative injury. Our results suggest that cells from Snell dwarf mice may use autophagy as a rescue mechanism to escape from cell death after exposure to oxidative stress.

Much remains to be done. Cells from Snell dwarf mice also show blunted responses of stress-induced ERK kinases, augmented induction of immediate early genes, higher levels of plasma membrane transport of reducing equivalents, resistance to oxygen-dependent growth crisis, and elevated expression of many genes regulated by nuclear factor (erythroid-derived2)-like2 (NFE2L2/NRF2). Developing and testing models that link these phenomena to one another, to autophagy control, and to underlying epigenetic changes may shed light on the ways in which early-life GH and IGF1 signals mold cell stress and set the pace of aging. It would also be very helpful to know which tissues in intact mice might show altered regulation of autophagy, in global and tissue-specific GHR mutants that have recently become available. Resolution of these questions may shed light on the cellular basis of disease resistance and longevity in long-lived mutants by manipulation of the GH-IGF1 signaling pathway. In addition, we have recently found that cells from long-lived species of rodents, and long-lived species of birds, are resistant to the lethal effects of oxidative and nonoxidative stresses in culture, and evaluation of species-specific differences in autophagy pathways may help to define how evolutionary changes in rate of aging are manifest at the level of cell biology.

Wang M, Miller RA. Fibroblasts from long-lived mutant mice exhibit increased autophagy and lower TOR activity after nutrient deprivation or oxidative stress. Aging Cell. 2012 doi: 10.1111/j.1474-9726.2012.00833.x.