SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging.

Genomic instability and alterations in gene expression are hallmarks of eukaryotic aging. The yeast histone deacetylase Sir2 silences transcription and stabilizes repetitive DNA, but during aging or in response to a DNA break, the Sir complex relocalizes to sites of genomic instability, resulting in the desilencing of genes that cause sterility, a characteristic of yeast aging. Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Neurotoxic and neurotrophic roles of proNGF and the receptor sortilin in the adult and ageing nervous system.

The precursor form of the nerve growth factor (proNGF), forms a heterotrimeric complex with the receptors p75 and sortilin; this complex has been implicated in neuron cell death. However, it is not known whether proNGF and the receptors p75 and sortilin contribute to age- and disease-related neurodegeneration. Here we show that proNGF induces cell death in subpopulations of basal forebrain and peripheral sympathetic neurons of old, but not of young, adult rodents. In contrast, proNGF appears to induce neurite outgrowth rather than cell death of young adult sympathetic neurons. We have examined the neurotoxic role of proNGF in old age, and find that proNGF protein is elevated during ageing in the projection areas of some populations of vulnerable central and peripheral neurons; caloric restriction, which has known neuroprotective effects, partially prevents these increases. Sortilin was found to play a significant part in the observed patterns of age-related proNGF-mediated neurotoxicity. In particular, survival of aged neurons was rescued by neurotensin, an alternative sortilin ligand that blocks the sortilin-mediated effects of proNGF. Furthermore, sortilin immunoreactivity increases markedly in ageing rodent basal forebrain and sympathetic neurons; in contrast, p75 levels are either unchanged or reduced. From these data we propose that selective age-related neuronal atrophy and neurodegeneration may be mediated by increased sortilin expression in neurons, together with elevated levels of proNGF expression in some targets.

ProNGF, sortilin, and age-related neurodegeneration.

Several studies have sought to demonstrate that neurodegeneration during disease and in old age is associated with reduced neurotrophic support. Little positive evidence has been forthcoming, either in relation to the availability of neurotrophins or to expression and function of the relevant receptors. Recently, a novel way in which neurotrophins could contribute to neurodegeneration has been suggested. In contrast to the well-known neurotrophic functions of the mature beta-form of nerve growth factor (mNGF), its precursor proNGF has recently been shown to be abundant in the adult brain and in the brains of patients with Alzheimer's disease. proNGF is synthesized as 25 and 32 kDa isoforms, which are glycosylated to form a principal 40 kDa species. Studies of the cortical targets of NGF-responsive basal forebrain neurons show that the 40 kDa form of proNGF is secreted in response to nerve stimulation, along with the proteases needed to generate the 13 kDa mNGF, or to degrade it. We have recently found that levels of 40 kDa proNGF are elevated in the aging brain and also in targets of peripheral NGF-responsive neurons. proNGF has been shown to be neurotoxic when bound in a heterotrimer with the p75 receptor and the receptor sortilin (identical to the neurotensin receptor NTS3). Interestingly, we find that sortilin levels increase in aged central and peripheral neurons, perhaps making these neurons more vulnerable to age-related increases in proNGF. Whether elevated levels of proNGF in targets or of sortilin in neurons contribute to known patterns of age- and disease-related neurodegeneration has not been previously investigated. Using in vitro models, our preliminary data now indicate that proNGF is indeed neurotoxic for aged, but not young, NGF-responsive basal forebrain and sympathetic neurons and that blockade of sortilin rescues proNGF-induced cell death. We therefore propose that increased proNGF in targets combined with increased sortilin expression in projecting neurons contributes to age-related neuronal atrophy and degeneration.

Prolyl isomerase Pin1 regulates neuronal differentiation via ß-catenin.

The Wnt/ß-catenin pathway promotes proliferation of neural progenitor cells (NPCs) at early stages and induces neuronal differentiation from NPCs at late stages, but the molecular mechanisms that control this stage-specific response are unclear. Pin1 is a prolyl isomerase that regulates cell signaling uniquely by controlling protein conformation after phosphorylation, but its role in neuronal differentiation is not known. Here we found that whereas Pin1 depletion suppresses neuronal differentiation, Pin1 overexpression enhances it, without any effects on gliogenesis from NPCs in vitro. Consequently, Pin1-null mice have significantly fewer upper layer neurons in the motor cortex and severely impaired motor activity during the neonatal stage. A proteomic approach identified ß-catenin as a major substrate for Pin1 in NPCs, in which Pin1 stabilizes ß-catenin. As a result, Pin1 knockout leads to reduced ß-catenin during differentiation but not proliferation of NPCs in developing brains. Importantly, defective neuronal differentiation in Pin1 knockout NPCs is fully rescued in vitro by overexpression of ß-catenin but not a ß-catenin mutant that fails to act as a Pin1 substrate. These results show that Pin1 is a novel regulator of NPC differentiation by acting on ß-catenin and provides a new postphosphorylation signaling mechanism to regulate developmental stage-specific functioning of ß-catenin signaling in neuronal differentiation.

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function.

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy.

Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues.

Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein.

Various functions are ascribed to the HBx regulatory protein of the hepatitis B virus (HBV). Due to the low expression level of HBx, it has been difficult to correlate spatial and temporal HBx expression levels with specific functions. Based on a novel cell-permeable peptide, known as the translocation motif (TLM), cell-permeable HBx fusion proteins were generated. The TLM-HBx fusion protein is rapidly internalized from the medium into almost all cells, whereas no significant internalization was seen with wild-type HBx. The major fraction of internalized HBx protein moves from the cytoplasm to the nucleus. The cytosolic fraction, however, activates c-RAF1/extracellular-signal-related kinase 2 signalling and causes activation of activator protein 1 (AP1) and nuclear factor-kappaB. The TLM-HBx protein rescues HBV gene expression from an activator-deficient HBV genome. These results indicate that cell-permeable regulatory proteins provide a novel, efficient tool for a clearly defined, dose-dependent analysis of regulatory protein function, without affecting the integrity of the cell, and can be used for the safe reconstitution of virus production from a regulatory-protein-deficient virus genome.