Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease

The greatest risk factor for Alzheimer's disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid beta (Aβ) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. Previously, we developed an adult-onset Drosophila model of AD. Mutant Aβ42 accumulation led to increased mortality and neuronal dysfunction in the adult flies. Furthermore, we showed that lithium reduced Aβ42 protein, but not mRNA, and was able to rescue Aβ42-induced toxicity. In the current study, we investigated the mechanism/s by which lithium modulates Aβ42 protein levels and Aβ42 induced toxicity in the fly model. We found that lithium caused a reduction in protein synthesis in Drosophila and hence the level of Aβ42. At both the low and high doses tested, lithium rescued the locomotory defects induced by Aβ42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. Our data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis

Ageing-associated changes in transcriptional elongation influence longevity

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss

Mutations in PINK1 and PARK2 cause autosomal recessive parkinsonism, a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons. To discover potential therapeutic pathways, we identified factors that genetically interact with Drosophila park and Pink1. We found that overexpression of the translation inhibitor Thor (4E-BP) can suppress all of the pathologic phenotypes, including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which could potently suppress pathology in Pink1 and park mutants. Rapamycin also ameliorated mitochondrial defects in cells from individuals with PARK2 mutations. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. We also found that loss of the Drosophila LRRK2 homolog activated 4E-BP and was also able to suppress Pink1 and park pathology. Thus, in conjunction with recent findings, our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism

Dietary regulation of hypodermal polyploidization in C. elegans

BACKGROUND: Dietary restriction (DR) results in increased longevity, reduced fecundity and reduced growth in many organisms. Though many studies have examined the effects of DR on longevity and fecundity, few have investigated the effects on growth. RESULTS: Here we use Caenorhabditis elegans to determine the mechanisms that regulate growth under DR. We show that rather than a reduction in cell number, decreased growth in wild type C. elegans under DR is correlated with lower levels of hypodermal polyploidization. We also show that mutants lacking wild type sensory ciliated neurons are small, exhibit hypo-polyploidization and more importantly, when grown under DR, reduce their levels of endoreduplication to a lesser extent than wild type, suggesting that these neurons are required for the regulation of hypodermal polyploidization in response to DR. Similarly, we also show that the cGMP-dependent protein kinase EGL-4 and the SMA/MAB signalling pathway regulate polyploidization under DR. CONCLUSION: We show C. elegans is capable of actively responding to food levels to regulate adult ploidy. We suggest this response is dependent on the SMA/MAB signalling pathway

Lowered Insulin Signalling Ameliorates Age-Related Sleep Fragmentation in <i>Drosophila</i>

<div><p>Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly <i>Drosophila</i>. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing <i>Drosophila</i>. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.</p></div

Rapamycin rescued age-related night sleep fragmentation in an S6K-dependent manner.

<p>(A) Rapamycin treatment (9 d) did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, age 10 d, <i>n</i> = 21/20 control/rapamycin). (B) Average activity count data (30 min bins) under 12∶12 h LD. (C) Acute rapamycin treatment of 45-d-old flies for 3 d did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, <i>n</i> = 64/64). Rapamycin-mediated night sleep, bout number, and bout length were independent of (D) 4E-BP (<i>n</i> = 19/19) and (E) reduced autophagy (a = <i>da-Gal4/UAS-ATG5-RNAi</i>, <i>n</i> = 20/17 or genetic controls c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21 and c2 = <i>UAS-ATG5-RNAi/+</i>, <i>n</i> = 23/19). Flies with reduced autophagy responded to rapamycin as controls in sleep (<i>p</i> = 0.81), bout number (<i>p</i> = 0.82), and night bout length (<i>p</i> = 0.42) (GLM). (F) Ubiquitous expression of constitutively active S6K blocked the rescue of night sleep fragmentation by rapamycin (c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21, and c2 = <i>UAS-S6K^STDETE/+</i>, <i>n = </i>20/18, <i>S6K = da-Gal4/UAS-S6K^STDETE</i>, <i>n = </i>20/17). Flies expressing a constitutively active form of S6K significantly differed from controls in the response to rapamycin (sleep <i>p</i> = 0.01, bout number <i>p</i> = 0.03, night bout length <i>p</i> = <0.0001, GLM). Kruskal Wallis test with Dunn's multiple comparisons of selected pairs. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. Day behaviours of (D–F) are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s006" target="_blank">Figure S6B–D</a>.</p

Reduced IIS causes day hyperactivity through increased octopaminergic signalling.

<p>(A) Two days feeding with mianserin hydrochloride (0.2 mg/ml) reverted the day activity phenotype of <i>dilp2-3,5</i> mutants (age 10 d), but not night activity, sleep, sleep bouts, and sleep bouts length (<i>w^Dah n = </i>17/24, <i>dilp2-3,5 n</i> = 17/27 +/− mianserin). GLM was used to determine significance of treatment by genotype interactions in sleep and activity behaviours on treatment with mianserin in controls and IIS mutants. Significant differences were seen in day activity (<i>p</i> = 0.0031), in day sleep (<i>p</i> = 0.0148), and day bout number (<i>p</i> = 0.002), but not in night behaviours (activity <i>p</i> = 0.31, sleep <i>p</i> = 0.49, bout number <i>p</i> = 0.72, night bout length <i>p</i> = 0.15). (B) Average activity count data (30 min bins) under 12∶12 h LD. (A) Kruskal Wallis test with Dunn's multiple comparison of selected pairs. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m.</p

<i>dfoxo</i> affected daytime activity and sleep phenotypes of <i>INR^DN</i> flies.

<p>Loss of <i>dfoxo</i> in <i>da-Gal4/UAS-INR^DN</i> flies but not in wild-type flies (age 20 d) decreased day activity but had no effect on night activity, had no significant effect on wakefulness (average activity per waking minute), increased day sleep duration but had no effect on night sleep duration, and reverted the low sleep bout phenotype of <i>da-Gal4/UAS-INR^DN</i> flies by day but not at night and increased night sleep bout duration. <i>dfoxo</i> indicates the <i>dfoxo^Δ94</i>allele (<i>n = </i>35 for all genotypes). Kruskal Wallis test with Dunn's multiple comparison test (selected pairs). ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. Independent experiments verifying activity and sleep phenotypes of <i>INR^DN;dfoxo</i> double mutants are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s003" target="_blank">Figure S3</a>.</p

Day hyperactivity of IIS mutants is dependent on the <i>AkhR</i>.

<p>(A) Loss of <i>AkhR</i> abrogated the day activity phenotype of <i>dilp2-3,5</i> mutants (age 15 d, <i>w^Dah n</i> = 18, <i>dilp2-3,5 n</i> = 15, <i>AkhR n</i> = 18, <i>AkhR dilp-3,5 n</i> = 17). GLM was used to determine significance of genotype by genotype interactions in sleep and activity behaviours on loss of <i>AkhR</i> in controls and <i>dilp2-3,5</i> mutants. Significant differences were seen in day activity (<i>p</i> = 0.0057) and day bout number (<i>p</i> = 0.044) but not in day sleep (<i>p</i> = 0.14) or night behaviours (activity <i>p</i> = 0.09, sleep <i>p</i> = 0.63, bout number <i>p</i> = 0.22, night bout length <i>p</i> = 0.067). Corresponding nighttime behaviours are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s004" target="_blank">Figure S4A</a>. (B) Two-day tolbutamide (1.35 mg/ml) treatment increased day activity of <i>w^Dah</i> flies. Lack of <i>dfoxo</i>, <i>AkhR</i>, or <i>dilp2-3,5</i> blocked the tolbutamide effect on day activity (nighttime behaviour shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s004" target="_blank">Figure S4B</a>) (age 15 d, <i>w^Dah n</i> = 51/47, <i>AkhR n</i> = 30/34, <i>dfoxo^Δ94 n</i> = 36/34, <i>dilp2-3,5 n</i> = 22/18, +/− tolbutamide). Analysis of genotype by treatment interactions (GLM) in sleep and activity behaviours on tolbutamide treatment in <i>IIS</i>, <i>AkhR</i>, and <i>dfoxo</i> mutants compared to controls showed day activity (<i>p</i> = 0.049), day sleep (<i>p</i> = <0.0001), and bout number (<i>p</i> = 0.008) were significantly different. However, no differences were seen in night behaviours (activity <i>p</i> = 0.58, sleep <i>p</i> = 0.89, bout number <i>p</i> = 0.28). (C) Mass spectrometry measurement of octopamine levels in head extracts (age 10 d, <i>w^Dah n</i> = 7, <i>dilp2-3,5 n</i> = 6, <i>AkhR n</i> = 6, <i>AkhR,dilp2-3,5 n</i> = 6). (A and B) Kruskal Wallis test with Dunn's multiple comparison test (selected pairs). (C) Mann–Whitney test. (C) One-way ANOVA with Bonferroni's multiple comparison test. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m.</p

Dopamine receptor mutants do not respond to rapamycin treatment.

<p>(A) <i>DopR1</i> mutants had similar activity and sleep features as rapamycin-fed flies. Behaviour of <i>DopR1</i> mutants was not affected by rapamycin feeding (age 10 d, <i>n</i> = 64 for all genotypes). Flies were fed with rapamycin for 9 d. (B) <i>dilp2-3,5</i>, <i>DopR1</i> mutants had similar activity and sleep features as <i>dilp2-3,5</i> mutants (<i>n = </i>64 for all genotypes). (C) QRT-PCR analysis of dopamine receptor (<i>DopR1</i>) expression normalized to <i>Rpl32</i> expression and controls in head extracts of <i>dilp2-3,5</i> mutants (age 10 d, <i>n</i> = 9) and <i>da-Gal4/UAS/INR^DN</i> flies and <i>da-Gal4/INR^DN;dfoxo</i> mutants (age 10 d, <i>n</i> = 3). (D) Mass spectrometry measurement of dopamine levels in head extracts of female flies (age 10 d, <i>n = </i>3). (E) QRT-PCR analysis of <i>DAT</i> expression, normalized to <i>Rpl32</i> expression (<i>n</i> = 9). (F) Behaviour of IIS mutants after short-term exposure (2 d) to the tyrosine hydroxylase inhibitor 3IY (5 mg/ml) (age 35 d, <i>n</i> = 32 for all genotypes). IIS mutants differed from controls in the nighttime activity and sleep response to 3IY treatment, but not in bout or bout length (activity <i>p</i> = 0.044, sleep <i>p</i> = 0.014, bout number <i>p</i> = 0.276, night bout length <i>p</i> = 0.463, GLM). (G) Behaviour of IIS mutants after short-term (12 h) exposure to METH (1 mg/ml) (age 25 d, <i>n</i> = 48 for all genotypes). IIS mutants differed from controls in daytime behaviours after METH treatment (activity <i>p</i> = 0.005, sleep <i>p</i> = <0.0001, bout number <i>p</i> = 0.0002, bout length <i>p</i> = 0.031, GLM) along with nighttime bouts (<i>p = </i><0.0001) and bout length (<i>p = </i><0.0001), whereas nighttime activity and sleep did not differ (activity <i>p = </i>0.796, sleep <i>p = </i>0.352, GLM). (A, B, G) Kruskal Wallis test with Dunn's multiple comparison of selected pairs. (F) Individual comparisons by Mann–Whitney U test. (C–E) Two-tailed <i>t</i> test. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. (C, E, F) QRT-PCR analysis normalized to <i>RNApolII</i> expression shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s005" target="_blank">Figure S5D</a>.</p