Elizabeth Fernandez

Rapamycin fed late in life extends lifespan in genetically heterogeneous mice

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.

Rapamycin, But Not Resveratrol or Simvastatin, Extends Life Span of Genetically Heterogeneous Mice

Rapamycin was administered in food to genetically heterogeneous mice from the age of 9 months and produced significant increases in life span, including maximum life span, at each of three test sites. Median survival was extended by an average of 10% in males and 18% in females. Rapamycin attenuated age-associated decline in spontaneous activity in males but not in females. Causes of death were similar in control and rapamycin-treated mice. Resveratrol (at 300 and 1200 ppm food) and simvastatin (12 and 120 ppm) did not have significant effects on survival in male or female mice. Further evaluation of rapamycins effects on mice is likely to help delineate the role of the mammalian target of rapamycin complexes in the regulation of aging rate and age-dependent diseases and may help to guide a search for drugs that retard some or all of the diseases of aging.

Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction.

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet‐restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin‐treated and diet‐restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.

WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

Parkinson’s disease (PD) is characterized by the progressive loss of nigrostriatal dopamine neurons leading to motor disturbances and cognitive impairment. Current pharmacotherapies relieve PD symptoms temporarily but fail to prevent or slow down the disease progression. In this study, we investigated the molecular mechanisms by which the non‐selective cannabinoid receptor agonist WIN55,212‐2 (WIN) protects mouse nigrostriatal neurons from 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced neurotoxicity and neuroinflammation. Stereological analyses showed that chronic treatment with WIN (4 mg/kg, intraperitoneal), initiated 24 h after MPTP administration, protected against MPTP‐induced loss of tyrosine hydroxylase‐positive neurons in the substantia nigra pars compacta independently of CB1 cannabinoid receptor activation. The neuroprotective effect of WIN was accompanied by increased dopamine and 3,4‐dihydroxyphenylacetic acid levels in the substantia nigra pars compacta and dorsal striatum of MPTP‐treated mice. At 3 days post‐MPTP, we found significant microglial activation and up‐regulation of CB2 cannabinoid receptors in the ventral midbrain. Treatment with WIN or the CB2 receptor agonist JWH015 (4 mg/kg, intraperitoneal) reduced MPTP‐induced microglial activation, whereas genetic ablation of CB2 receptors exacerbated MPTP systemic toxicity. Furthermore, chronic WIN reversed MPTP‐associated motor deficits, as revealed by the analysis of forepaw step width and percentage of faults using the inverted grid test. In conclusion, our data indicate that agonism at CB2 cannabinoid receptors protects against MPTP‐induced nigrostriatal degeneration by inhibiting microglial activation/infiltration and suggest that CB2 receptors represent a new therapeutic target to slow the degenerative process occurring in PD.

Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males

Four agents — acarbose (ACA), 17‐α‐estradiol (EST), nordihydroguaiaretic acid (NDGA), and methylene blue (MB) — were evaluated for lifespan effects in genetically heterogeneous mice tested at three sites. Acarbose increased male median lifespan by 22% (P < 0.0001), but increased female median lifespan by only 5% (P = 0.01). This sexual dimorphism in ACA lifespan effect could not be explained by differences in effects on weight. Maximum lifespan (90th percentile) increased 11% (P < 0.001) in males and 9% (P = 0.001) in females. EST increased male median lifespan by 12% (P = 0.002), but did not lead to a significant effect on maximum lifespan. The benefits of EST were much stronger at one test site than at the other two and were not explained by effects on body weight. EST did not alter female lifespan. NDGA increased male median lifespan by 8–10% at three different doses, with P‐values ranging from 0.04 to 0.005. Females did not show a lifespan benefit from NDGA, even at a dose that produced blood levels similar to those in males, which did show a strong lifespan benefit. MB did not alter median lifespan of males or females, but did produce a small, statistically significant (6%, P = 0.004) increase in female maximum lifespan. These results provide new pharmacological models for exploring processes that regulate the timing of aging and late‐life diseases, and in particular for testing hypotheses about sexual dimorphism in aging and health.

Evaluation of Resveratrol, Green Tea Extract, Curcumin, Oxaloacetic Acid, and Medium-Chain Triglyceride Oil on Life Span of Genetically Heterogeneous Mice

The National Institute on Aging Interventions Testing Program (ITP) was established to evaluate agents that are hypothesized to increase life span and/or health span in genetically heterogeneous mice. Each compound is tested in parallel at three test sites. It is the goal of the ITP to publish all results, negative or positive. We report here on the results of lifelong treatment of mice, beginning at 4 months of age, with each of five agents, that is, green tea extract (GTE), curcumin, oxaloacetic acid, medium-chain triglyceride oil, and resveratrol, on the life span of genetically heterogeneous mice. Each agent was administered beginning at 4 months of age. None of these five agents had a statistically significant effect on life span of male or female mice, by log-rank test, at the concentrations tested, although a secondary analysis suggested that GTE might diminish the risk of midlife deaths in females only.

Neurodegeneration and Motor Dysfunction in Mice Lacking Cytosolic and Mitochondrial Aldehyde Dehydrogenases: Implications for Parkinson's Disease

Previous studies have reported elevated levels of biogenic aldehydes in the brains of patients with Parkinsons disease (PD). In the brain, aldehydes are primarily detoxified by aldehyde dehydrogenases (ALDH). Reduced ALDH1 expression in surviving midbrain dopamine neurons has been reported in brains of patients who died with PD. In addition, impaired complex I activity, which is well documented in PD, reduces the availability of the NAD+ co-factor required by multiple ALDH isoforms to catalyze the removal of biogenic aldehydes. We hypothesized that chronically decreased function of multiple aldehyde dehydrogenases consequent to exposure to environmental toxins and/or reduced ALDH expression, plays an important role in the pathophysiology of PD. To address this hypothesis, we generated mice null for Aldh1a1 and Aldh2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Aldh1a1−/−×Aldh2−/− mice exhibited age-dependent deficits in motor performance assessed by gait analysis and by performance on an accelerating rotarod. Intraperitoneal administration of L-DOPA plus benserazide alleviated the deficits in motor performance. We observed a significant loss of neurons immunoreactive for tyrosine hydroxylase (TH) in the substantia nigra and a reduction of dopamine and metabolites in the striatum of Aldh1a1−/−×Aldh2−/− mice. We also observed significant increases in biogenic aldehydes reported to be neurotoxic, including 4-hydroxynonenal (4-HNE) and the aldehyde intermediate of dopamine metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL). These results support the hypothesis that impaired detoxification of biogenic aldehydes may be important in the pathophysiology of PD and suggest that Aldh1a1−/−×Aldh2−/− mice may be a useful animal model of PD.

Interaction of a glucocorticoid-responsive element with regulatory sequences in the promoter region of the mouse tyrosine hydroxylase gene

The purpose of the work reported here was to determine whether the tyrosine hydroxylase glucocorticoid‐responsive element (TH‐GRE) interacts with the cyclic AMP pathway and the CRE in regulating mouse TH promoter activity, and whether an additional, previously identified downstream GRE‐like element also participates in the function of the TH‐GRE and CRE. To determine the role of the cAMP pathway on TH‐GRE function, we compared the effects of forskolin and dexamethasone on TH mRNA, TH gene transcription and TH promoter activity in a mutant PC12 cell line (A126–1B2) deficient in cAMP‐dependent protein kinase A (PKA) with their effects in the wild‐type parental strain. Forskolin treatment increased TH mRNA content, transcriptional activity and the activity of a chimeric gene with 3.6 kb of the TH promoter in wild‐type cells, but not in PKA‐deficient cells. In contrast, dexamethasone treatment stimulated equivalent increases in TH mRNA, TH gene transcription and TH promoter activity in each cell type. Mutation of the CRE in chimeric constructs containing 3.6 kb of the 5′ flanking sequence of the mouse TH gene or coexpression of a dominant‐negative mutant of CREB prevented the stimulation of TH promoter activity by forskolin. However, neither the mutation of the CRE nor inhibition of CREB influenced basal or dexamethasone‐stimulated promoter activity. Site‐directed mutagenesis of the TH‐GRE eliminated the response of the promoter to dexamethasone. However, the mutagenesis of a more proximal 15‐bp region with a GRE‐like sequence had no demonstrable effect on the ability of dexamethasone to stimulate TH promoter activity. Neither mutagenesis of the TH‐GRE or the downstream GRE‐like sequence had an effect on the ability of forskolin to activate this chimeric gene. Taken together, these results provide evidence that a single GRE is sufficient for maximal induction of transcriptional activity by glucocorticoids and that the CRE is not required for either partial or full activity of this upstream GRE sequence.

Transcriptional and post-transcriptional regulation of Tyrosine Hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C -dependent pathways

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Protein Synthesis Blockade Differentially Affects the Degradation of Constitutive and Nicotinic Receptor‐Induced Tyrosine Hydroxylase Protein Level in Isolated Bovine Chromaffin Cells

Abstract: Continuous incubation of bovine adrenal chromaffin cells with the nicotinic receptor agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) causes a twofold increase in the steady‐state level of catalytically active tyrosine hydroxylase (TH) protein by 3‐4 days. The present study examined the processes that control the time course of enzyme induction. In cells exposed to DMPP for 36 or 54 h, incorporation of [3H]leucine into TH was increased 1.9‐ and 2.2‐fold, respectively, compared with control (non‐DMPP‐treated) cells. The increase correlated with a twofold rise in TH mRNA level, indicating the absence of translational control of TH synthesis by DMPP. Also absent was an effect by DMPP on the rate of degradation of TH protein because pulse‐chase analysis estimated a half‐life for TH of 26 ± 5 h in DMPP‐treated cells, a value that was (a) essentially the same as that estimated in control cells (29 ± 3 h), (b) within the same range as that estimated by approach to steady state (t1/2 = 19 ± 4 h), which measured the decline of TH protein content from the DMPP‐induced steady‐state level back to the basal value during deinduction with the nicotinic antagonist hexamethonium, and (c) consistent with the time course of accumulation of TH protein to a new steady‐state level in response to DMPP. However, different rates of degradation for TH protein were observed in control and DMPP‐treated cells under conditions in which protein synthesis was blocked. In control cells incubated with 100 μM puromycin or 20 μM cycloheximide for 3 days, the level of catalytically active TH protein failed to decline and exhibited a half‐life of ≥250 h. This finding indicated that TH protein was stabilized. TH protein level also failed to decline when cells were incubated for 3 days with a concentration of the transcription inhibitor α‐amanitin that caused a >90% loss of TH mRNA. Thus, degradation of constitutively expressed TH protein appears to be controlled by processes dependent on ongoing transcription and translation. In contrast, the increased amount of TH induced by DMPP was not stabilized but instead underwent a decline to the basal level following addition of puromycin or cycloheximide. It is important to note, however, that the decline occurred at a slower rate (t1/2≥ 45 h) than that measured during deinduction. Taken together, these data suggest that alterations in the rate of degradation of TH protein may play a role in controlling TH level when TH synthesis is blocked but not when TH synthesis is increased, such as during nicotinic receptor stimulation.