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.2019 Oct;574(7778):359-364.
doi: 10.1038/s41586-019-1647-8. Epub 2019 Oct 16.

Regulation of lifespan by neural excitation and REST

Affiliations

Regulation of lifespan by neural excitation and REST

Joseph M Zullo et al. Nature.2019 Oct.

Abstract

The mechanisms that extend lifespan in humans are poorly understood. Here we show that extended longevity in humans is associated with a distinct transcriptome signature in the cerebral cortex that is characterized by downregulation of genes related to neural excitation and synaptic function. In Caenorhabditis elegans, neural excitation increases with age and inhibition of excitation globally, or in glutamatergic or cholinergic neurons, increases longevity. Furthermore, longevity is dynamically regulated by the excitatory-inhibitory balance of neural circuits. The transcription factor REST is upregulated in humans with extended longevity and represses excitation-related genes. Notably, REST-deficient mice exhibit increased cortical activity and neuronal excitability during ageing. Similarly, loss-of-function mutations in the C. elegans REST orthologue genes spr-3 and spr-4 elevate neural excitation and reduce the lifespan of long-lived daf-2 mutants. In wild-type worms, overexpression of spr-4 suppresses excitation and extends lifespan. REST, SPR-3, SPR-4 and reduced excitation activate the longevity-associated transcription factors FOXO1 and DAF-16 in mammals and worms, respectively. These findings reveal a conserved mechanism of ageing that is mediated by neural circuit activity and regulated by REST.

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Figures

Extended Data Figure 1.
Extended Data Figure 1.. Partitioning of the aging human population for analysis of gene expression in the brain.
a-c, Adjusted gene expression profiles forage-associated genes were compared between cognitively normal agedindividuals to derive a matrix of Pearson correlation coefficients thatindicate the degree of similarity between any two cases in the ROSMAP (a,dorsolateral prefrontal cortex, n=150 individuals) CommonMind Consortium (b,dorsolateral prefrontal cortex, n=174 individuals) and Gibbs (c, frontalcortex, n=40 individuals) cohorts.d-f, Most significantlyenriched GO terms for upregulated genes in the cortex of cognitively normalindividuals who lived to be ≥85 years old relative to individuals wholived to be ≤80 years old in the ROSMAP (d, n=117 individuals),CommonMind Consortium (e, n=155 individuals), and Gibbs (f, n=37individuals) cohorts. P-values were calculated using Fisher’s exacttest (see Methods).g,Meta-analysis of GO term enrichment for downregulated genes. Shown areselected GO terms related to excitatory and inhibitory synaptictransmission. The individual cohort enrichment p-values were combined usingStouffer’s method (see Methods).NS, FDR>0.1.
Extended Data Figure 2.
Extended Data Figure 2.. Ivermectin and nemadipine extend lifespan without interfering with worm motility.
a, Worms were transferred at day 8 to either standardNGM plates or plates containing ivermectin (1 pg/ml) or nemadipine (2μM). Shown is a representative curve of an experiment repeated twice.Nemadipine versus WT: P=3.2 e-4; Ivermectin versus WT: P=2.2e-7 by log-ranktest. Nemadipine, n=81; Ivermectin, n=82; WT, n=76.b. Day 2worms treated with nemadipine or ivermectin for 24 hours were transferred toliquid culture and thrashing rate was assessed using the NemametrixwMicrotracker (see Methods). Shown aremean motility scores for the first 60 minutes, ± S.E.M.. Untreated,n=17 wells; Ivermectin, n=17 wells; Nemadipine, n=16 wells. Each wellcontained ~10 worms. **P=1.7e-4 vs untreated, Mann–Whitney Utest with multiple testing correction by Holm’s method. Results arerepresentative of an experiment replicated twice.
Extended Data Figure 3.
Extended Data Figure 3.. Repression of multiple neurotransmitter systems extends lifespan inC. elegans.
a-h.C. elegans lines expressing the transgenic HisCl1 channelin the indicated neuronal populations were treated with 10 mM histamine(His+) starting at adult day 1 (a, c, e, g) or day 8 (b, d, f, h) andcompared to untreated controls (His−). P-values are by log-rank. SeeSupplementary Table22 for individual n values and statistics.i, Shownis the mean lifespan extension ± S.E.M. for worms treated withhistamine at days 1 or 8 relative to untreated controls for at least threeindependent replicates, *P<0.05, **P<0.01 by Student’st-test. HisCl1was driven using the GAL4SK:VP64 systemfor the GABAergic (GABA), glutamatergic (GLUT) and cholinergic systems,usingunc-47,eat-4, andunc-17 drivers, respectively (see Supplementary Table 19 fordetails).j, Reduced ASH neuron excitation following inhibitionof GABA activity at day 1 but not day 8. Shown is normalized maximum GCaMPfluorescence in day 1 and 8unc-47:HisCl1 worms that weretreated with 10 mM histamine [His (+)] on the indicated day, or untreatedcontrols [His (−)]. Day 1 His(−): n=18 worms; Day 1 His(+),n=19 worms; Day 8 His(−): n=23 worms; Day 8 His(+): n=20 worms.*P=1.1e-3 by the Mann–Whitney U test.
Extended Data Figure 4.
Extended Data Figure 4.. Neural excitation, neuropeptide signaling and lifespan inC. elegans.
a, Increased excitation of ASH neurons following RNAiagainst the GABA vesicular transporterunc-47. GCaMPimaging was performed on worms with enhanced neuronal RNAi (See Figure 3 legend and Methods for details) forunc-47 (n=37)or controls (n=43) at day 2. **P= 6.8e-3 by the Mann–Whitney U test.b, RNAi forunc-47 reduces lifespan. Wormswith enhanced neuronal RNAi were treated withunc-47 (n=31)or control RNAi (n=84). Shown is a representative lifespan analysisreplicated 3 times. P= 1.3e-6 by log-rank test.c, Reduction ofsynaptic neurotransmission or neuropeptide signaling extend lifespan inC. elegans. Mutations in genes affecting glutamatergicneurotransmission (eat-4), presynaptic function(unc-13) and neuropeptide signaling(egl-3) exhibit comparable lifespan extension. WT,n=57;eat-4(nj2), n=54. P≤2.2e-16;unc-13(e51), n=92, P=3.6e-14;egl-3(gk238), n=35, P=8.3e-11 bylog-rank test. Shown are curves representative of two independentreplicates.d, Extension of lifespan byegl-3RNAi in worms with enhanced neuronal RNAi. Shown are lifespan curvesrepresentative of two independent replicates.egl-3 RNAi(n=47 worms); Empty Vector (n=84 worms). P=3.5e-11 by the log-rank test.
Extended Data Figure 5.
Extended Data Figure 5.. Gene regulation and neural activity associated with REST and extended longevity.
a-b, Expression of genes downregulated in individuals≥85 years versus ≤80 years old is inversely related to RESTmRNA levels. Shown is linear regression analysis of normalized and adjustedREST mRNA levels and mean expression ofa, all downregulatedgenes andb, downregulated genes associated with the synaptictransmission GO term. Data is from the CommonMind cohort. Each pointrepresents an individual case, n=155 individuals. P-values were derived by at-test for the slope of the regression line. Note similarity to the data forthe ROSMAP cohort in Fig. 2a, b.c-d, Stratification byage group. Analysis of the ROSMAP cohort (c, n=117 individuals) and theCommonMind cohort (d, n=155 individuals) as in Fig 2a, but stratified by age group. P-values were derived byt-test for the slope of the regression line.e, Loss of RESTexpression in conditional REST knockout mice. Representative images of thecortex (top panel) and hippocampus (bottom panel) from RESTlx/lx(Control), and Nestin-Cre;RESTlx/lx(REST−/−) mice. Immunolabeling was performed with theanti-mouse REST-14 antibody directed against the REST C-terminal domain.Scale bar, 40 μm. Image shown is representative of an experimentreplicated 4 times.f, Survival of REST −/− andcontrol mice following administration of the seizure-inducing agentpentylentetrazole (PTZ, 40 mg/kg). P=0.065 for REST−/− versuscontrol by the log-rank test. Control, n=9; REST−/−, n=7.
Extended Data Figure 6.
Extended Data Figure 6.. Induction ofspr-4 extends lifespan and suppresses neural excitation inC. elegans.
a,spr-4 mRNA levels in worms expressing a stably integrateddCas9::VP64 transgene in the presence, sgRNA(+), or absence sgRNA(−),of 4 different sgRNAs targeting thespr-4 promoter.Transcript levels were determined by qRT-PCR and normalized tosgRNA(−) controls. Values are the mean ± S.E.M., n=3. A: *P=0.041; B: P=0.020 by one-sided Student’st-test.b, dCas9::VP64-mediated elevation of SPR-4 protein levels.Left panel: Representative images of the head region of heterozygous F1progeny of the strains bearing apspr-4::spr-4::gfp::spr-4utr transgene. Arrowheadsindicate SPR-4::GFP positive nuclei. Dashed red lines indicate the outlineof the worm body. Scale bar, 40 μm. Middle panel: Values representthe mean ± S.E.M. sgRNA(−), n=5 worms; sgRNA(+), n=5 wormswith 7-38 measurements per worm; *P=0.022, one-sided Student’st-test. Right panel: Values represent the mean ± S.E.M.sgRNA(−), n=4 worms; sgRNA(+), n=4 worms. P=0.011, one-sidedStudent’s t-test. Shown is a representative experiment replicatedthree times.c, Extended lifespan in worms expressing anintegrated dCas9::VP64 transgene and sgRNAs targeting thespr-4 promoter [sgRNA(+)] (n=79 worms) relative todCas9::VP64-expressing worms in the absence of sgRNAs [sgRNA (−)](n=57 worms) P=5.5e-9, log-rank test. Representative of an experimentreplicated 6 times.d, Lifespans of worms expressing sgRNAtargeting thespr-4 promoter in the presence (n=87 worms)or absence (n=58 worms) of dCas9::VP64. P=3.7e-7, log-rank test.Representative of an experiment replicated twice.e, Lifespansof dCas9::VP64 expressing worms in the presence (n=51 worms) or absence(n=58 worms) of sgRNAs on thespr-4(tm465) loss-of-functionmutant background. P=0.49, log-rank test. Representative of threeindependent replicates.f, Overexpression ofspr-4 reduces neural excitation. GCaMP imaging wasperformed in ASH neurons in SPR-4 overexpressing (sgRNA+) and control(sgRNA-) worms in the lines described inc. Shown are maximumGCaMP fluorescence changes. sgRNA minus, n=12 worms; sgRNA plus, n=10 worms.*P=0.025, Mann–Whitney U test.
Extended Data Figure 7.
Extended Data Figure 7.. Lifespan extension byspr-4 overexpression and inhibition of neural excitation aredaf-16-dependent.
a, Lifespan extension by overexpression ofspr-4 isdaf-16 dependent. Lifespansof worms overexpressingspr-4 (sgRNA+;dCAS9::VP64) or notoverexpressingspr-4 (sgRNA-;dCAS9::VP64) followingtreatment withdaf-16 RNAi or an empty vector control.sgRNA(+)EV (n=29 worms) versus sgRNA(−)EV (n=25 worms): P=2.7e-4;sgRNA(+)daf-16 (n=18 worms) versussgRNA(−)daf-16 (n=29 worms) P=0.20 by log-ranktest. Representative of 4 independent replicates.b, c,Lifespan extension by the neural excitation inhibitors ivermectin andnemadipine isdaf-16-dependent. Shown are lifespandeterminations for WT control anddaf-16(mu86) mutant worms in thepresence or absence of nemadipine (2μM) (b) orivermectin (1pg/ml) (c).b,WT, n=69 worms;WT+Nema, n=51;daf-16, n=43;daf-16+Nema,n=67. WT+Nema vs WT, P=9.9e-8; daf-16+Nema vs daf-16, P=0.014 by log-ranktestc, WT, n=78 worms; WT+Ive, n=77;daf-16,n=27;daf-16+Ive, n=29. WT+Ive vs WT, P=7.3e-8;daf-16+Ive vsdaf-16, P=0.22; log-ranktest. Curves are representative of an experiment replicated 2 (nemadipine)or 3 (ivermectin) times.d, Inhibition of neural excitationwith ivermectin elevates DAF-16 levels. Worms expressing a DAF-16::GFPtransgene were treated for 10 days with 1 pg/mL ivermectin and assessed byconfocal microscopy. Left panel: Shown is total DAF-16::GFP. Valuesrepresent the mean ± S.E.M (untreated, n=19 worms, Ivermectin, n=16worms). **P=2.5e-7, Mann-Whitney U test. Right panel: Shown is nuclearDAF-16::GFP (n=5 worms per group, 50-61 nuclei per worm). *P= 0.013 byStudent’s t-test. Results are representative of an experimentreplicated twice.e, DAF-16 is not required for inhibition ofneural excitation by nemadipine. Shown are maximum ASH GCaMP intensitychanges for day 2daf-16(mu86) mutantworms treated for 24 hours with 2 μM nemadipine (untreated, n=16worms; nemadipine, n=18 worms). P=9.4e-5, Mann-Whitney U test).f, DAF-16 is not required for inhibition of neuralexcitation by ivermectin. Shown are day 2 worms treated for 24 hours with 1pg/mL ivermectin (control, n=19 worms; ivermectin, n=32 worms). P=0.030,Mann-Whitney U test).
Extended Data Figure 8.
Extended Data Figure 8.. SPR-3 and SPR-4 contribute to lifespan extension and gene regulation associated with reduced DAF-2 insulin/IGF-like signaling.
a, Loss of function of SPR-3 and SPR-4 reduces thelifespan extension ofdaf-2 RNAi. Left panel:Representative lifespan analysis ofspr-4(by105);spr-3(ok2525) double mutant and wild-type(WT) worms followingdaf-2 or empty vector (EV) controlRNAi. WT+EV, n=54 worms;spr-4;3+EV, n=58 worms;WT+daf-2, n=26 worms;spr-4;3+daf-2, n=54 worms. Rightpanel: Values represent mean percent lifespan extension ± S.E.M.(daf-2 RNAi versus EV control) in the indicatedgenotypes. WT, n=6 independent experiments;spr-4(by105), n=3, *P=0.017 versus WT;spr-4(tm465), n=4, **P=0.0062 versusWT;spr-3(ok2525), n=4, **P=0.0018 versusWT;spr-4(by105);spr-3(ok2525),n=4, **P=0.0016 versus WT by Studentst-test. See Supplementary Table22 for individual lifespan data and statistics.b,Lifespan is unaffected byspr-4 andspr-3mutations in a wild-type background. WT, n=50 worms;spr-3(ok2525), n=31;spr-4(by105);spr-3(ok2525),n=32;spr-4(by105), n=34;spr-4(tm465), n=33.There were noreproducibly significant changes by the log-rank test in 3-6 independentexperiments per genotype (see Supplementary Table 22).c, Quantification of lifespan extension indaf-2 mutant worms shown in Fig. 3b attributable to neuronal expression ofspr-3 andspr-4. RNAi was targeted toneurons by neuronal expression of asid-1 transgene inotherwisesid-1 nulldaf-2(1370) mutants(daf-2;p[neuron]:sid-1), and comparedwith untargeted RNAi insid-1 wild-typedaf-2(1370) mutants (daf-2). Valuesrepresent mean lifespan extension relative to the controlsid-1(pk3321);p[neuron]:sid-1worms treated with empty vector (EV) ± S.E.M (n= 3 independentexperiments). Significant lifespan effects were not observed for RNAi in theabsence of thedaf-2 mutation. *P<0.05;**P<0.01 by Student’st-test.d,Gene expression determined by RNA sequencing in day 2 adult worms.Differentially expressed genes (rows) and the indicated worm genotypes(columns) were clustered, and gene expression, transformed to a z-score pergene, is represented in a heat map. N=3 independent replicates per genotype.e, Venn diagram illustrating the overlap of differentiallyexpressed genes indaf-2 single mutant vs WT andspr-4;3;daf-2 triple mutant vsdaf-2single mutant comparisons. P=7e-30, Fisher’s exact test with aone-sided alternative hypothesis.. f, Long-liveddaf-2 mutants do not show an age-related increase inneural excitation. Shown is maximum ASH GCaMP intensity changes in day1-2(n=39) and day 14-16 (n=20)daf-2(e1370) mutant worms. Notethe absence of the age-related increase in excitation observed in wild-typeaging worms (Fig. 1e). P=0.93,Mann-Whitney U test.g, Thespr-4;spr-3 doublemutant in a wild-type background does not significantly affect neuralexcitation in ASH neurons. WT, n=15 worms;spr-4;spr-3,n=15 worms. P= 0.62, Mann-Whitney U test.h, DAF-16 does notmediate suppression of neural excitation in thedaf-2mutant. RNAi fordaf-16 was performed indaf-2(e1370) mutant worms on asid-1(pk3321);p[neuron]:sid-1background to augment RNAi in neurons (daf-16 RNAi, n=20worms, empty vector (EV) control, n=12 worms). P=0.33, Mann–Whitney Utest.i, Description of the genes targeted by RNAi in Figure 4d.
Extended Data Figure 9.
Extended Data Figure 9.. Regulation of DAF-16 by SPR-3 and SPR-4.
a, Reduced DAF-16 activation inspr-4;3 mutants followingdaf-2 RNAi.Left confocal panel: Shown are day 10 worms of the indicated genotypesexpressing an integrated DAF-16::GFP transgene and treated withdaf-2 RNAi or empty vector (EV) control since day 1 ofadulthood. Images are maximum intensity z-projections. Scale bar,40μm. Left bar graph: Values represent mean GFP intensity ±S.E.M. in the peri-pharyngeal regions ofspr-4;3 doublemutants relative to wild-type controls for a representative experimentreplicated 4 times (see methods fordetails of analysis). (n=8-12 worms per replicate). **P=5.2e-5 byWelch’s t-test. Right confocal panel: Higher magnification views ofDAF-16::GFP and DAPI-labeled nuclei. Images are magnified confocal z-planes.Scale bar, 10μm. Right bar graph: Values represent mean nuclear GFPintensity ± S.E.M. relative to the WT-EV control, n=5 worms pergenotype and 51-89 nuclei per worm. *P=0.016, **P=5.5e-3 by ANOVA withpost-hoc Tukey test. Values and images are representative of an experimentreplicated 3 times.b, Gene expression determined by RNAsequencing in adult day 10 worms. Differentially expressed genes (rows) andreplicates of the indicated worm genotypes (columns) were clustered, andgene expression, transformed to a z-score per gene, is represented in a heatmap. n=3 independent replicates per genotype.c, Venn diagramillustrating the overlap of differentially expressed genes in day 10daf-2 vs WT andspr-4;3;daf-2 vsdaf-2 comparisons. P=4e-123, Fisher’s exact testwith a one-sided alternative hypothesis.d, Overlap of class Idaf-16 target genes (described in Methods) with genes downregulated in day 10spr-4;3;daf-2 triple mutants relative todaf-2 single mutants. P-values were calculated using ahypergeometric distribution (see Methods). n.s, p=0.99e, Ivermectin increasesDAF-16::GFP levels inspr-4;3 worms followingdaf-2 RNAi. Left panel: Confocal imaging of GFPfluorescence in ivermectin-treated (10 pg/ml) and untreated worms. The reddashed lines indicate the worm body. Right panel: Quantification ofDAF-16::GFP. Values represent mean GFP intensity ± S.E.M.,WT/Untreated, n=12; WT/Ivermectin, n=10;spr-4;3/Untreated,n=10;spr-4;3/Ivermectin, n=10. **P=4.6e-4(spr-4;3 vs WT untreated), P=2.6e-4(spr-4;3 +Ivermectin vsspr-4;3untreated) by Mann–Whitney U test with multiple testing correction byHolm’s method. Shown is a representative experiment replicated 3times.
Extended Data Figure 10.
Extended Data Figure 10.. Coregulation of FOXO1 and REST in the aging brain and modulation by glutamatergic signaling.
a, Linear regression analysis of REST and FOXO mRNAlevels in the prefrontal cortex of 174 cognitively-intact individuals(age≥60 years) from the CommonMind Consortium determined by RNAsequencing. P-values are derived from at-test for theslope of the regression line and Bonferroni-corrected across all expressedgenes.b, Colocalization of REST and FOXO1 in neurons of theaged human prefrontal cortex. Confocal immunofluorescence microscopy wasperformed in human prefrontal cortex using antibodies against REST (green,rabbit polyclonal; Bethyl), FOXO1 (red, goat polyclonal; LS-Bio) and theneuronal marker MAP2 (grey, chicken polyclonal; Abcam). Scale bar, 40μm. The image shown is representative of immunofluorescence labelingperformed in 30 individuals. See Supplementary Table 20 foradditional information on antibodies.c, Inhibition ofglutamatergic signaling in mouse cortical neuronal cultures elevates FOXO1levels. Left panel: Primary mouse cortical neuronal cultures treated withkynurenic acid (KYNA, 5 μM), APV (50 μM), NBQX (2 μM)or vehicle (Control) were analyzed by confocal immunofluorescence for FOXO1or MAP2 and labeled with DAPI. Boxed areas were magnified in the lower threerows. Note that most FOXO1 in cultured neurons is cytoplasmic, but adetectable nuclear component overlaps with DAPI. Scale bar, 40 μm.Right panel: Quantification of total and nuclear FOXO1 levels inMAP2-positive neurons. Values represent the mean ± S.E.M. Control,n=200; KYNA, n=326; APV, n=148; NBQX, n=197. FOXO1 Total/KYNA: **P=2.1e-8;FOXO1 Nuclear/KYNA **P=1.1e-4; FOXO1 Total/NBQX **P=8.8e-13; FOXO1nuclear/NBQX **P=5.2e-6 by the Mann–Whitney U test with multipletesting correction by Holm’s method. Shown is a representativeexperiment replicated 3 times.
Figure 1.
Figure 1.
Neural excitation and longevity in humans andC.elegans.a, Analysis of the cortical transcriptomeprofile in cognitively intact aged individuals from the ROSMAP cohort.Unsupervised hierarchical clustering shows a transcriptional signature of down-and up-regulated genes associated with extended longevity.b-d,Most significantly enriched gene ontology (GO) terms for downregulated genesassociated with extended longevity (≥85 versus ≤80 years of age)in the ROSMAP (dorsolateral prefrontal cortex, n=117) (b), CommonMind Consortium(dorsolateral prefrontal cortex, n=155) (c), and Gibbs (frontal cortex, n=37)(d) cohorts. P-values were calculated by Fisher’s exact test (see Methods).e, AgingC.elegans exhibit increased neuronal excitation. Shown are themaximum GCaMP fluorescence intensity changes in ASH neurons of young adult (day1-2) and older (day 12-16) worms. Young, n=82 worms; Old, n=30 worms. *P=3.6e-4by Mann–Whitney U test.f, The L-type calcium channelblocker nemadipine (2μM) represses neural excitation. Control, n=14;Nemadipine, n=13. *P=0.029, Mann–Whitney U test.g,Nemadipine extends lifespan. Worms were continuously treated with 2 μMnemadipine beginning at adult day 1, P=7.7e-11, log-rank test. Control, n=59;Nemadipine, n=50, replicated 3 times.h, The chloride channelagonist ivermectin (1pg/ml) reduces neural excitation. Control, n=18;Ivermectin, n=23. *P=0.038, Mann–Whitney U test.i,Extension of lifespan by continuous treatment with ivermectin beginning at adultday 1 (Control, n=35; 0.01 pg/ml: n=34, P= 0.62 ; 0.1 pg/ml: n=33, P= 1.5e-3;1pg/ml: n=42, P= 1.9e-3, log-rank test), replicated 3 times. Summary statisticsfor all individual lifespan experiments are in Supplementary Table 22.
Figure 2.
Figure 2.
REST regulates neural excitation in the aging brain and is associatedwith extended longevity.a-b, Expression of genes downregulated inindividuals with extended longevity (≥85 versus ≤80 years old) isinversely related to REST mRNA levels. Shown is linear regression analysis ofthe mean expression ofa, all downregulated genes andb, downregulated genes associated with the synaptictransmission GO term. Data is from the ROSMAP cohort. Each point represents anindividual case (n=117). P-values were derived by t-tests of the regression lineslopes.c, Increased nuclear REST levels in the prefrontal cortexof centenarians. Left panel: Immunofluorescence labeling for REST (green, rabbitpolyclonal; Bethyl laboratories) and DAPI (blue) in human prefrontal cortex.Scale bar, 40 μm. Right panel: Nuclear REST levels in cognitively intactindividuals 70-80 years (n=9) and >100 years (n=7) of age. Valuesrepresent the mean ± S.E.M, **P=1.5e-4, Student’st-test.d, REST represses neural excitation inthe mouse cerebral cortex. Shown are images from PET-CT scanning offluorodeoxyglucose (18F-FDG) uptake in 18-month-oldNestin-Cre;RESTlx/lx (REST−/−) and age-matchedRESTlx/lx (Control) mice.e, Average standardizeduptake value (SUV) at increasing time intervals after injection of18F-FDG. Values represent the mean ± S.E.M., n=7 mice pergroup. *P<0.05, **P<0.01, Mann-Whitney U test.f,Increased epileptiform discharges in aged REST-deficient mice. Upper Panel: EEGrecording from REST(−/−) and age-matched control mice. LowerPanel: Number of mice with at least one epileptiform discharge (≥ 3 secs)in a 48 hour recording. Control, n=9; REST−/−, n=7. *P=0.035,Fisher’s exact test.g, Seizure duration afteradministration of PTZ (40 mg/kg). Control, n=6; REST −/−, n=6mice. *P=0.016, Mann–Whitney U test.
Figure 3.
Figure 3.
C. elegans REST orthologs mediate longevity indaf-2 loss-of-function mutants.a, The RESTorthologs spr-4 andspr-3 are required formaximal longevity indaf-2 mutant worms. Lifespan analysis wasperformed on wild-type anddaf-2(1370) loss-of-function mutantworms, and the indicated combinations ofdaf-2 andspr-4/spr-3 mutations. Thespr-4/spr-3mutations significantly reduced the lifespan ofdaf-2 mutantworms. n=29-59 worms per genotype, replicated at least three times per genotype.P<0.001 for all curves relative todaf-2, by log-ranktest.b, Neuronal expression ofspr-3 andspr-4 mediate lifespan extension indaf-2mutant worms. Shown are lifespans of worms with neuronal targeting of RNAi byneuronal expression of asid-1 transgene in otherwisesid-1 nulldaf-2(1370) mutants, oruntargeted RNAi insid-1 wild-typedaf-2(1370)mutants. Lifespan effect of neuronal targeting ofspr-4;3 RNAiversus EV control RNAi is significant by log rank test (P=2.5e-6), n=22-56 wormsper curve replicated at least 4 times.c, SPR-3 and SPR-4 repressgenes that mediate neural excitation. Shown are significantly enriched GO termsfor upregulated genes related to neural excitation in RNA-seq analysis of day 2spr-4;3;daf-2 triple mutants versusdaf-2single mutant worms. P-values were calculated using Fisher’s exact test(see Methods), n=3 biological replicatesper genotype.
Figure 4.
Figure 4.
SPR-3 and SPR-4 suppress multiple neurotransmitter and neuropeptidesystems to extend lifespan indaf-2 mutant worms.a, Neural excitation is suppressed indaf-2mutants and partially restored byspr-4;3 mutations. GCaMPimaging was performed in ASH neurons. Shown is the fraction of worms with atleast 1 firing event in a 2 minute recording. Values represent the mean ±S.E.M., n= 4-5 independent experiments. **P=7.9e-07 (daf-2 vsWT), P=1.5e-4 (spr-4;3;daf-2 vsdaf-2); P=0.0011(spr-4;3;daf-2 vs WT) by ANOVA with post-hoc Tukeytest.b, Quantification of GCaMP fluorescence changes in day 2worms: WT, n=53;daf-2, n=25;spr-4;3;daf-2,n=26. **P=3.1e-6 (daf-2 vs WT), P=1.5e-3(spr-4;3;daf-2 vsdaf-2); *P=0.018(spr-4;3;daf-2 vs WT) ,Mann–Whitney U test with multiple testing correction by Holm’smethod.c, Inhibition of neural excitation by ivermectin (+Ive, 10pg/ml) reverses lifespan reduction byspr-4;3 mutations indaf-2 mutant worms (P=1.1e-16spr-4;3;daf-2 +Ive versus-Ive)daf-2-Ive, n=53 worms;daf-2+Ive, n=55;spr-4;3;daf-2-Ive, n=95;spr-4;3;daf-2+Ive, n=69;WT-Ive, n=64.d, Multiple neurotransmitter and neuropeptide signaling systemscontribute to the effects ofspr-4;3 mutants on longevity.Change in lifespan ofspr-4;3;daf-2 triple mutant anddaf-2 single mutant worms following neuronal RNAi for theindicated genes relative to empty vector control RNA. RNAi was targeted toneurons as described in Fig. 3b.*P<0.05, **P<0.01, Student’st-test. n=3independent experiments per group. Individual statistics are in Supplementary Table 22.
Figure 5.
Figure 5.
REST regulates FOXO1 expression in the mammalian brain.a, Linear regression analysis of REST and FOXO1 mRNAexpression in the prefrontal cortex of cognitively intact individuals (ROSMAPcohort age 71-101 years, n=150) determined by RNA sequencing. P-values werederived by linear regression t-tests for the slope with Bonferroni correctionfor all expressed genes.b, Coordinate regulation of REST and FOXO1in human prefrontal cortex. Nuclear REST and FOXO1 protein levels weredetermined by immunofluorescence microscopy in pyramidal neurons of theprefrontal cortex in individual young adult (20-38 yrs), aged (70-80 yrs), andcentenarian (>100 yrs) cases. Each point represents a neurondouble-labeled for REST and FOXO1. n=71-114 neurons per individual, P-valueswere derived as ina.c, FOXO1 induction in the agingmouse cortex is REST-dependent. Left panel: Immunocytochemical labeling forFOXO1 and the neuronal marker MAP2 in cortical neurons of RESTlx/lx(Control) and Nestin-Cre;RESTlx/lx (REST−/−) mice at 9and 18 months of age. Scale bar is 40 μm. Right panel: Quantitation ofFOXO1 nuclear levels. Values represent the mean ± S.E.M. Control 9 mo: n= 4 mice; Control 18 mo: n = 9 mice; REST−/− 9 mo: n =4 mice; REST−/− 18 mo: n = 5 mice. **P=1.2e-5 cntrl 9mo vs cntrl 18mo; **P=4.4e-7 cntrl 18 mo vs REST−/− 18 mo by ANOVAwith post-hoc Tukey test.
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