REST and stress resistance in ageing and Alzheimer’s disease

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An Addendum to this article was published on 16 November 2016

Abstract

Human neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer’s disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer’s disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid β-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer’s disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.

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Figure 1: Induction of REST in the ageing human prefrontal cortex.
Figure 2: Regulation of REST target genes in ageing and AD.
Figure 3: REST is neuroprotective.
Figure 4: Caenorhabditis elegans SPR-4 protects against oxidative stress and Aβ toxicity.
Figure 5: REST, autophagy and proteostasis in AD, DLB and FTD.
Figure 6: Nuclear REST is positively correlated with cognitive function and longevity.

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Primary accessions

Gene Expression Omnibus

Data deposits

Data are available in the Gene Expression Omnibus (GEO) under accession GSE53890.

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Acknowledgements

We thank members of the Yankner laboratory for suggestions and discussion, Monlan Yuan, Allison Harwick, Kelly Dakin and Gregory Klein for assistance, and Cheng Li and Dana Gabuzda for helpful discussion. We also acknowledge the Rush Alzheimer's Disease Center, the Brigham and Women’s Hospital Brain Bank, the Massachusetts General Hospital ADRC Brain Bank, and the Kathleen Price Bryan Brain Bank at Duke University for providing tissue samples. This work was supported by an NIH Director’s Pioneer Award (DP1OD006849) and NIH grants PO1AG27916 and RO1AG26651 to B.A.Y., RO1GM072551 to M.P.C., P30AG10161, R01AG15819 and R01AG17917 to D.A.B., and a grant from the Glenn Foundation for Medical Research to B.A.Y. J.Z. is a Molecular Biology of Neurodegeneration fellow at Harvard Medical School.

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Authors

Contributions

T.L., L.A., J.Z., Y.P., H.K., and H.-M.K. performed experiments. T.L., L.A., J.Z., M.C. and B.A.Y. contributed to the overall study design. T.L., Y.C., T.-H.Y., D.D. and X.L. performed informatics analysis. D.A.B. contributed tissue samples, cognitive test data and analysis. B.A.Y. directed the study and B.A.Y., T.L., L.A. and J.Z. wrote the manuscript, which was edited by all the coauthors.

Corresponding author

Correspondence to Bruce A. Yankner.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Neuroanatomical distribution of REST induction in the ageing human brain.

a, Representative confocal photomicrographs of hippocampal CA1 pyramidal neurons (top), dentate gyrus granule neurons (middle, DG) and cerebellar Purkinje and granule cell neurons (bottom), from young, aged or AD cases that were labelled for REST (green). Note the increased expression of REST in hippocampal CA1 pyramidal and dentate granule cell neurons in aged cases relative to young adult cases. Also note that nuclear REST is markedly reduced in AD in hippocampal CA1 neurons, but not in dentate granule cell or cerebellar neurons. b, Specificity of REST immunohistochemistry. Shown are sections of normal aged PFC labelled with anti-REST (Bethyl) (upper panel), anti-REST preincubated with a REST blocking peptide (middle panel), and non-specific IgG (lower panel). REST labelling is green; nuclear labelling with DAPI is blue. c, REST knockdown (REST shRNA) and overexpression (REST-OE) in SH-SY5Y cells confirm antibody specificity. Also shown is labelling with antibody preincubated with REST blocking peptide (REST-OE+blocking). REST knockdown and overexpression were confirmed by western blotting (Extended Data Fig. 3b). d, Quantification of nuclear REST levels in hippocampal CA1, CA3, CA4 regions, in dentate gyrus granule cells (DG), and in cerebellar Purkinje cell neurons. REST is induced with age in CA1, CA3, CA4 and DG neurons, but not in cerebellar neurons. REST expression is reduced in AD in CA1, CA3, and CA4 neurons, but not in DG or cerebellar neurons. CA1: young n = 11, aged n = 30, AD n = 33. CA3, CA4, DG and cerebellum: young n = 8–9, aged n = 7–8, AD = 9–10. Values are the mean ± s.e.m., *P < 0.05, **P < 0.01 and ***P < 0.001 by Student’s unpaired t-test. Scale bars, 25 µm.

Extended Data Figure 2 ChIP-seq analysis of REST target genes shows enrichment for genes related to cell death and the pathology of AD.

a, Genes identified by REST ChIP-seq in SH-SY5Y cells are downregulated in the ageing human PFC. Expression of age-regulated REST target genes identified by ChIP-seq shows a highly significant inverse correlation with age at death. For each case, a weighted expression index was derived for age-regulated target genes based on microarray analysis. These values were normalized to the youngest adult value (24 years; 100%). n = 43, age range 24–106 years. b, Canonical pathways by Ingenuity IPA analysis of REST ChiP-seq targets. c, REST ChIP-seq binding peaks in genes related to cell death pathways and AD pathology. d, Confirmation of ChIP-seq targets by quantitative ChIP-PCR following REST overexpression (OE-REST) or REST knockdown (sh-RESTa and sh-RESTb) in SH-SY5Y cells. Shown is ChIP followed by real time PCR using primers that amplify REST binding sites identified by ChIP-seq. PCR amplification of a region of the actin promoter not proximal to a known RE1 site was used as a negative control. Values are normalized to the input control and represent the mean ± s.e.m., n = 3. *P < 0.05 relative to control by Student’s unpaired t-test.

Extended Data Figure 3 REST regulates expression of genes related to cell death and the neuropathology of AD.

a, REST knockdown with either of two distinct shRNAs (sh-RESTa and sh-RESTb) significantly increases mRNA expression of ChIP-seq targets related to cell death and AD pathology, whereas REST overexpression (REST) represses gene expression. Values are the mean ± s.d., n = 3. *P < 0.05 by Student’s unpaired t-test. b, Shown are western blots of lysates from SH-SY5Y cells transduced with a control lentiviral vector (CTRL), or 3 separate REST shRNAs (sh-REST; each lane represents a different shRNA) or REST cDNA (REST) to knockdown or overexpress REST, respectively. c, REST knockdown (sh-REST) markedly induces phosho-tau epitopes (PHF-1 and AT8). Induction of these epitopes is blocked by lithium chloride (LiCl), an inhibitor of the tau kinase GSK3β.

Extended Data Figure 4 REST protects against oxidative stress.

a, Cortical neuronal cultures from REST-deficient (REST cKO) mice show extensive neuritic degeneration relative to control cultures after incubation with 5 µM oligomeric Aβ42 for 24 h, which is prevented by lentiviral transduction of REST (REST cKO + REST). Neuritic processes are labelled with antibody TUJ1. Scale bar, 30 µm. b, Pro-apoptotic genes show increased mRNA expression in REST-deficient cortical neuronal cultures after treatment with hydrogen peroxide (50 µM H2O2, 3 h) or oligomeric Aβ42 (5 µM, 8 h). Increased expression was reversed by lentiviral transduction of REST (REST cKO+REST). Values are the mean ± s.e.m., n = 4. *P < 0.05 by Student’s unpaired t-test. c, REST knockdown potentiates hydrogen peroxide-induced apoptosis in SH-SY5Y cells. Increased cell death was prevented by transducing a REST construct (mouse REST) resistant to the shRNA. Cells were incubated with 800 µM H2O2 for 2 h, and apoptotic cells were quantified by FACS analysis of annexin V–APC. Values represent fold change relative to the untreated control. d, REST knockdown (Sh-REST) increases vulnerability to oxidative stress in cultured primary human cortical neurons treated with 100 µM H2O2 for 2 h relative to control cultures (Sh-CTRL). e, REST knockdown increases generation of reactive oxygen species (ROS). Shown are FACS profiles of a fluorescent ROS indicator (CellRox). Note right-ward shift (increased ROS) in H2O2-treated (600 µM, 2 h) SH-SY5Y cells (red) versus untreated controls (blue) following lentiviral transduction of REST shRNA (Sh-REST). f, Increased ROS levels in SH-SY5Y cells expressing either of two different REST shRNAs (sh-RESTa and sh-RESTb) is reversed by the antioxidant N-acetyl cysteine (NAC, 5 mM). g, REST protects against oxidative DNA damage. Shown is a measure of DNA fragmentation in the comet assay following treatment of SH-SY5Y cells with H2O2. Note increase in DNA damage induced by either of two REST shRNAs (Sh-RESTa and Sh-RESTb) relative to cells transduced with a control shRNA (Sh-CTRL). Conversely, overexpression of REST reduces oxidative DNA damage relative to overexpression of GFP (CTRL). Horizontal bars indicate the mean value. **P < 0.0001 by the Mann–Whitney test; n = 114–134. Values in b, c, d and f represent the mean ± s.e.m, n = 6–10, *P < 0.05, **P < 0.01 by Student’s unpaired t-test. h, Inverse correlation between oxidative DNA damage and nuclear REST levels in normal ageing and early AD (AD1). Shown is double-labelling for REST and 8-oxoguanine (8oxoG) in hippocampal CA1 neurons. Scale bar, 20 µm.

Extended Data Figure 5 Caenorhabditis elegans spr genes and oxidative stress resistance.

a, Mutations of spr-1, spr-3 and spr-4 reduce survival in the presence of oxidative stress. Worms were continuously incubated with the superoxide-generating agent paraquat (5 mM). Shown are representative time courses of survival, and quantification of reduced mean lifespan relative to wild-type in worms incubated with paraquat. Shown are mutants in spr-1 and spr-3, two different mutants in spr-4, and a double spr-3/spr-4 mutant (spr-4(by105);spr-3(ok2525)). The spr-4(by105);spr-3(ok2525) mutant showed similar survival to wild-type in the absence of paraquat. Values represent the decrease in mean lifespan as percentage relative to wild type and represent the mean ± s.d., n = 3. *P < 0.05 by log-rank test. b, Depletion of spr-4 by RNAi increases sensitivity to oxidative stress and phenocopies the spr-4(by105) mutation. Worms were fed RNAi against the indicated genes or an empty vector control and then transferred to plates seeded with standard OP50 bacteria and containing 5 mM paraquat. Shown is the percent decrease in mean survival relative to the empty vector control from 3 independent experiments. Two spr-4 RNAi-expressing bacterial strains were used (Methods). In addition to N2, we also used a C. elegans strain (TU3270) with enhanced dsRNA uptake in neurons (Methods). One of the spr-4 RNAi strains generated a significantly greater reduction in survival in the TU3270 background compared with N2. The paraquat sensitivity of worms fed RNAi against the antioxidant gene sod-1 was used as a positive control. Values are the mean ± s.d., n = 3. *P < 0.05 by log-rank test. c, A stably integrated SPR-4::GFP construct under the control of the endogenous spr-4 promoter is expressed predominantly in neurons in adult worms, as indicated by colocalization with the neuronal marker prab-3::mCherry. Top, pharyngeal ring neurons; bottom, tail neurons. d, Treatment of adult worms with paraquat (+PQ) from day 1 to day 4 induces expression of SPR-4::GFP. Untreated worms (-PQ). Upper panels show confocal imaging of a representative strain; the lower panel graph shows quantitative analysis of SPR4::GFP expression in 3 separate strains. Horizontal bars indicate the median; boxed areas represent the second and third quartiles. n = 3 (15 worms each); ***P < 0.001 by unpaired t-test. e, REST represses expression of the presenilin hop-1 in C. elegans. Expression of hop-1 mRNA was measured by qRT–PCR in 24-h post L4 worms of the indicated genotypes. For each replicate, transcript values were normalized to cdc-42. Note that hop-1 mRNA expression is increased in the spr-4(by105) mutant, and that repression is partially restored by wild-type spr-4 or human REST (REST). Values represent fold change relative to the wild-type control (1) and represent the mean ± s.d., n = 3. *P < 0.05 by Student’s t-test. Scale bars, 20 µm.

Extended Data Figure 6 Induction of REST by stress and cell non-autonomous signalling.

a–d, Redox-active Fe+2 (15 µM), Aβ42 (15 µM), H2O2 (indicated concentrations), and the glutathione synthesis inhibitor BSO (50 µM) increase REST protein levels in primary human cortical neuronal cultures. Shown are western blots for REST and actin. e, REST mRNA levels determined by qRT–PCR. f, Treatment of human neurons with Fe+2 or Aβ42 increase REST–RE1 site binding in the SNAP25 and calbindin 1 genes as determined by ChIP-qPCR. g, REST mRNA levels measured by qRT–PCR after addition of conditioned medium from H2O2-treated (30 µM, H30; 100 µM, H100; 300 µM, H300; 600 µM, H600; 800 µM, H800) or control neuronal cultures (CTRL CM) to naive neurons. h, Extracts from aged human PFC (O) induce REST when added to SH-SY5Y cells. Much lower levels of induction are induced by extracts derived from young adult (Y) or AD cortex. Shown are western blots for REST, β-catenin and β-actin. Values in eg represent the mean ± s.d., n = 3. *P < 0.05, **P < 0.01 by Student’s unpaired t-test.

Extended Data Figure 7 Induction of REST by Wnt signalling.

a, Cell non-autonomous induction of REST by aged brain extracts is partially inhibited by the Wnt antagonist Dickkopf (DKK). Extracts were derived from young adult (Y), aged (O) or AD PFC of the indicated ages and then incubated with SH-SY5Y cells in the absence (-) or presence (+) of DKK (250 ng ml−1). b, Conditioned medium transferred from SH-SY5Y cells, treated with either wortmannin (2 µM), tunicamycin (2 µM), H2O2 (100 µM) or LiCl (10 mM), induce REST when added to naive cells, which is inhibited by DKK. c, REST levels are increased by treatment of SH-SY5Y cells with Wnt3a or 7a (250 ng ml−1, 16 h; upper western blot), and by LiCl (5 or 10 mM, 6 h) or Chiron 99021 (20 nM, 6 h) (lower western blot). d, REST ChIP-seq shows that the Wnt activator LiCl (5 mM) broadly increases REST binding to target genes. Shown are REST targets (P < 10−5) with REST–RE1 site binding within 10 kb of the transcription start site. They include genes related to AD pathology, such as the gamma secretase presenilin 2 (PSEN2), and pro-apoptotic genes (PUMA, BAX, DAXX, TRADD and BCL2L11). e, f, SH-SY5Y cells incubated with LiCl (10 mM, 24 h) or Chiron 99021 (100 nM, 24 h) exhibit increased nuclear REST. Nuclei are stained dark blue with DAPI, which become light blue when there is overlap with the green REST staining (e). Quantitative analysis of per cent REST-positive nuclei (f). g, Increased nuclear β-catenin in the PFC in normal ageing, and reduced levels in AD. Shown is FACS analysis of neuronal nuclei isolated from PFC. Values are the mean ± s.e.m. *P < 0.05, **P < 0.01 by Student’s unpaired t-test. Young, n = 13; Aged, n = 18; AD, n = 10. h, Colocalization of REST and β-catenin in the nucleus of ageing neurons in the PFC. Aged and AD cases were double-labelled for REST (green) and β-catenin (red). Scale bars, 20 µm.

Extended Data Figure 8 Autophagy and REST.

a, REST is distributed in a vesicular punctate distribution in the cytoplasm of cortical neurons in AD. Confocal immunofluorescence microscopy shows colocalization of REST with the autophagosome markers LC3, Atg7 and Atg12, but not with the lysosomal markers LAMP1 or LAMP2. b, Nuclear REST levels are reduced by activation of autophagy. SH-SY5Y cells were subject to serum withdrawal (-FBS, 40 h) to induce autophagy and maintained in Opti-MEM to preserve cell viability. Note that serum withdrawal results in depletion of nuclear REST (loss of green labelling that overlaps with the blue DAPI nuclear stain), which is restored by bafilomycin (-FBS/Baf). c, The autophagy inhibitors 3-methyladenine (3-MA, 5 mM) and bafilomycin (Baf, 150 nM) increase nuclear REST. Values represent per cent of cells positive for nuclear REST, and represent the mean ± s.e.m., n = 3; **P < 0.01 by Student’s t-test. Scale bars, 15 µm.

Extended Data Figure 9 Neuronal REST levels are positively correlated with cognitive function and inversely correlated with AD neuropathology.

a, Linear regression analysis shows that nuclear REST levels in PFC neurons are positively correlated with measures of cognition function. REST levels were determined by FACS analysis of isolated PFC neuronal nuclei. Each point represents an individual case, and REST level is normalized as fold change relative to the mean value of the young adult group. n = 37, age range 67–90 years. b, Nuclear REST levels in prefrontal cortical neurons decrease with increasing AD pathology. Shown are CERAD (1-frequent plaques, 2–3 sparse/moderate plaques, 4-no plaques) and NIA-Reagan scores (composite index of neuritic plaques and neurofibrillary tangles; 1, high likelihood of AD; 2, intermediate likelihood; 3, low likelihood). Values are the mean ± s.e.m. CERAD 1, n = 20; 2–3, n = 11; 4, n = 9; NIA-Reagan 1, n = 12; 2, n = 14; 3, n = 14. *P < 0.05, ***P < 0.001.

Extended Data Figure 10 REST, longevity and stress resistance.

a, Prefrontal cortical (upper panel), hippocampal CA1 (middle panel) and cerebellar (lower panel) sections of individuals ranging in age from 57–102 years were double-labelled for REST (green) and the neuronal marker MAP2 (red). Cases with extreme longevity (98, 102 years) are associated with marked induction of REST in prefrontal cortical and hippocampal CA1 neurons, but not in cerebellar Purkinje cell (ovals) or granule cell neurons (to the right of the dashed line). Scale bar, 20 µm. b, REST increases the expression of genes associated with stress resistance and longevity. Shown are fold changes in mRNA levels for catalase (CAT), superoxide dismutase (SOD1) and FOXO1 following REST knockdown (sh-RESTa or sh-RESTb) or overexpression (REST). Values represent the mean ± s.d., *P < 0.05 by Student’s unpaired t-test. n = 3. c, FOXO1 expression is dependent on REST. Western blotting of SH-SY5Y cells shows that shRNA-mediated REST knockdown (sh-REST +) almost completely abolishes FOXO1 protein, which is prevented by shRNA-resistant mouse REST.

Supplementary information

Supplementary Table 1

Human Brain Specimens. This table provides demographic, diagnostic, physical and source data for human brain specimens used in the study. (XLS 97 kb)

Supplementary Table 2

Primers for PCR and ChIP. This table provides sequence, amplicon and accession number data on PCR primers used in the study. (XLS 56 kb)

Supplementary Table 3

This table lists Antibodies and Reagents. This file was replaced on 16 November 2016 - see http://www.nature.com/doifinder/10.1038/nature20579 for details. (XLS 41 kb)

Supplementary Table 4a

ChIP-seq Data: SH-SY5Y Cells. This table provides data on locations of binding sites, binding fold changes relative to input controls, statistical significance and RE1 motif analysis. (XLS 546 kb)

Supplementary Table 4b

ChIP-seq Data: SH-SY5Y Cells/Lithium Chloride. This table provides the same categories of information as Table 4a but for SH-SY5Y cells that were treated with lithium chloride. (XLS 743 kb)

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Lu, T., Aron, L., Zullo, J. et al. REST and stress resistance in ageing and Alzheimer’s disease. Nature 507, 448–454 (2014). https://doi.org/10.1038/nature13163

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