SUPPLEMENTARY INFORMATION

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Jordan Wilkins
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1 doi:1.138/nture1849 neurotoxic insults cute synptic dysfunctions chronic cognitive deficits epigenetic lockde of gene trnscription e.g. Bdnf IV, synptophysin neurotoxic insults Aβ H 2 O 2 Cdk5/p25 P GR1 P GR1 GRE Hdc2 other Supplementry Figure 1. Model of the potentil consequences nd cuses of elevted levels in the neurodegenerting rin., Neurotoxic insults re known to cutely constrin synptic functions, which in turn, cn led to more chronic cognitive dysfunctions. Here, we propose tht neurotoxic insults cn directly led to cognitive dysfunctions y epigeneticlly silencing neuroplsticity genes vi the mechnisms descried elow., Upon neurotoxic insults such s extrcellulr Aβ-firils, H2O2, intrcellulr ccumultion of p25 nd/or other stimuli, GR1 ecomes phosphorylted on S211, mrk for its ctivtion. Phoshorylted GR1 inds to its responsive element (GR responsive element, GRE) in the proximl Hdc2 promoter region in the neurodegenerting mouse rin, nd stimultes the expression of Hdc2. In the presence of Aβ-firils nd H2O2, this stimultion is further potentited, ut olished when S211 is mutted to A211 (not shown). Elevted protein levels ind to the promoter region of lerning, memory nd synptic plsticity-relted genes such s BdnfIV, synptophysin nd others. There, inding co-occurs with reduced histone cetyltion, nd thus more compcted chromtin, preventing RNA polymerse from inding (not shown), supposedly the driving force ehind the reduced gene expression oserved. Such decresed expression of neuroplsticity genes is correlted with reduced synptic plsticity nd poor memory performnce in mice with neurodegenertion. 1

2 ntiody specificity DAPI -/- pgr1 ntiody specificity pgr1 DAPI Mock CIP Supplementry Figure 2. Specificity of nd pgr1 ntiody signls., Immunohistochemicl imges showing detection in the hippocmpus of wild-type mice, ut not in -/- mice. Scle r upper pnel, 8µm, lower pnel, 1µm., Immunocytochemicl imges showing pgr1 immunorectivity in mock-treted, ut not in clf intestinl phosphtse (CIP)-treted primry hippocmpl neurons (DIV14). Scle r, 1µm. 2

3 Dentte Gyrus CA3 DAPI DAPI c Amygdl DAPI Supplementry Figure 3. The elevtion of is restricted to re CA1 of the hippocmpus, nd further not oserved in the mygdl.,, Representtive immunohistochemicl imges of levels in the () dentte gyrus nd () hippocmpl re CA3 of mice compred to control littermtes. c, Representtive immunohistochemicl imges of levels in the mygdl of mice compred to control littermtes; scle r, 1µm (, ), 2µm (c). 3

4 GFP DAPI protein level (fold chnge of control) c GFP HDAC1 DAPI d HDAC1 protein level (fold chnge of control) e GFP HDAC3 DAPI f HDAC3 protein level (fold chnge of control) Supplementry Figure 4., ut not HDAC1 nd HDAC3, is incresed in the neurodegenerting cortex of mice., c, e, Representtive immunohistochemicl imges depicting (), (c) HDAC1, nd (e) HDAC3 levels in the prefrontl cortex of mice nd control littermtes (n=3-6 sections from 3 mice ech)., d, f, Quntifiction of (, c, e). Scle r, 2μm. p 1; vlues re men ± s.e.m. 4

5XFAD DAPI MERGE protein level (fold chnge of control) 5XFAD c HDAC1 DAPI MERGE d 5XFAD HDAC1 protein level

HDAC1 protein level (fold chnge of control) Supplementry Figure 5.

, c, Representtive immunohistochemicl imges depicting () nd (c) HDAC1 in the hippocmpus of 6-month-old 5XFAD

e, g, Representtive immunohistochemicl imges depicting (e) nd (g) HDAC1 in the prefrontl cortex of

5 5XFAD DAPI MERGE protein level (fold chnge of control) 5XFAD c HDAC1 DAPI MERGE d 5XFAD HDAC1 protein level (fold chnge of control) e DAPI MERGE f 5XFAD protein level (fold chnge of control) g HDAC1 DAPI MERGE h 5XFAD HDAC1 protein level (fold chnge of control) Supplementry Figure 5., ut not HDAC1 levels re incresed in the neurodegenerting forerin of 5XFAD mice., c, Representtive immunohistochemicl imges depicting () nd (c) HDAC1 in the hippocmpus of 6-month-old 5XFAD mice compred to control littermtes (n=3-6 sections from 3-4 mice ech)., d, Quntittive ssessment of () nd (c). e, g, Representtive immunohistochemicl imges depicting (e) nd (g) HDAC1 in the prefrontl cortex of 6-month-old 5XFAD mice compred to control littermtes (n=3-4 sections from 3 mice ech). f, h, Quntittive ssessment of (e) nd (g). Scle r, 5μm. p 5; vlues re men ± s.e.m. 5

6 HDAC1 ChIP (fold chnge of control) c d e HDAC3 ChIP (fold chnge of control) AcH2BK5 ChIP (fold chnge of control) AcH3K14 ChIP (fold chnge of control) AcH4K5 ChIP (fold chnge of control) HDAC1 inding 5. Lerning nd Memory 5. Synptic Plsticity 5. Housekeeping HDAC3 inding 5. H2BK5 cetyltion H3K14 cetyltion H4K5 cetyltion Arc Bdnf I Bdnf II Bdnf IV Cdk5 Egr1 Homer1 5. GluR1 GluR2 Nfl NR2A NR2B Syp Syt1 5. β-actin β-gloin β-tuulin Supplementry Figure 6. HDAC1 nd HDAC3 inding to neuroplsticity genes is overll not incresed in the hippocmpus of mice, ut the reduction of histone cetyltion in the promoter region of neuroplsticity genes is not restricted to H4K12.,, Quntittive PCR results of () HDAC1- nd () HDAC3-immunoprecipitted chromtin in the hippocmpus of mice versus control littermtes (n=4-6 mice ech). c-e, Quntittive PCR results of (c) AcH2BK5-, (d) AcH3K14- nd (e) AcH4K5-immunoprecipitted chromtin of the hippocmpus of mice versus control littermtes (n=3-8 nimls ech). p 5; p 1; p 1; vlues re men ± s.e.m. 6

7 inding to coding sequences ChIP (fold chnge of control) Arc Bdnf Cdk5 Egr1 Homer1 GluR1 GluR2 Nfl NR2A NR2B Syp Syt1 β-actin β-gloin β-tuulin Supplementry Figure 7. inding to the coding region of neuroplsticity genes is incresed in the hippocmpus of mice. Quntittive PCR results of - immunoprecipitted chromtin in the coding sequence of neuroplsticity genes in the hippocmpus of mice versus control littermtes (n=4-5 nimls ech). p 5; vlues re men ± s.e.m. 7

Ac-Tu Totl Tu Ac-p53 Totl p53 Ac-tuulin Protein level (fold chnge of control) Totl tuulin Ac-Tu/Tu Ac-p53/p53 Ac-tuulin/tuulin c Cytoplsm d Nucleus Ac-K Ac-K β-actin β-actin Supplementry Figure 8.

, Representtive imges of generl cetyl western lot nlysis of immunoprecipitted protein complexes with tu, p53 nd tuulin (n=3-6 mice ech); note tht the increse in tu nd p53 protein levels in the mice

8 Ac-Tu Totl Tu Ac-p53 Totl p53 Ac-tuulin Protein level (fold chnge of control) Totl tuulin Ac-Tu/Tu Ac-p53/p53 Ac-tuulin/tuulin c Cytoplsm d Nucleus Ac-K Ac-K β-actin β-actin Supplementry Figure 8. The elevtion of does not induce generl cetyltion chnges on proteins other thn histones., Representtive imges of generl cetyl western lot nlysis of immunoprecipitted protein complexes with tu, p53 nd tuulin (n=3-6 mice ech); note tht the increse in tu nd p53 protein levels in the mice hs een previously descried y our l, nd tht p53 ws found to e hypercetylted on lysine 382 (Kim et l., 27)., Quntifiction of (). c, d, Representtive imges of western lot nlysis of protein cetyltion in cytoplsmic (c) nd nucler (d) hippocmpl extrcts of mice nd control littermtes (n=3 mice ech); vlues re men ± s.e.m. 8

9 IP: WB: CoREST WB: LSD1 WB: SIN3A WB: MTA2 WB: IgG Input IgG Input Protein level (fold chnge of control) Protein level (fold chnge of control) CoREST SIN3A Protein level (fold chnge of control) Protein level (fold chnge of control) 4 LSD MTA Supplementry Figure 9. is more undntly ound to co-repressor complexes in the hippocmpus of mice., Representtive imges of western lot nlysis of protein levels of CoREST nd LSD1, memers of the CoREST complex, of SIN3A, memer of the Sin3 complex, nd of MTA2, memer of the NuRD complex, following immunoprecipittion of hippocmpl extrcts with (n=3-4 nimls ech)., Quntifiction of (). Protein levels were normlized to input, p 5; p 1; p 1; vlues re men ± s.e.m. 9

TROL shrna 2-1 shrna 2-2 shrna 2-3 shrna 2-4 α-tuulin ITR CMKII mcherry U6 shrna ITR c shrna 2-1 shrna 2-4 β-tuulin d protein level (fold chnge of control) shrna 2-1 shrna 2-4 e shrna 2-1 shrna 2-4

Figure 1. Strtegy to reduce expression y RNA interference.

A significnt downregultion ws oserved for constructs shrna2-1 nd shrna2-4. These constructs nd the scrmle shrna were then sucloned into the AAV vector under ().

10 TROL shrna 2-1 shrna 2-2 shrna 2-3 shrna 2-4 α-tuulin ITR CMKII mcherry U6 shrna ITR c shrna 2-1 shrna 2-4 β-tuulin d protein level (fold chnge of control) shrna 2-1 shrna 2-4 e shrna 2-1 shrna 2-4 mcherry DAPI f protein level (fold chnge of control) 2-1 untrnsfected shrna 2-4 g p25 induction stereotxic injection ehviorl testing, smple collection h 2 6 weeks, scr, scr, sh mcherry Supplementry Figure 1. Strtegy to reduce expression y RNA interference., Vlidtion of the trget specificity of different shrna clones s determined y lentivirl trnsduction in primry corticl neurons (DIV7) y western lot nlysis. A significnt downregultion ws oserved for constructs shrna2-1 nd shrna2-4. These constructs nd the scrmle shrna were then sucloned into the AAV vector under ()., Schemtic of the construct used for AAV production; shrna signifies either control scrmle shrna, or shrna constructs trgeting. c, Representtive imges of western lot nlysis of in primry hippocmpl neurons (DIV14) following trnsfection with the AAV-shRNA constructs. d, Quntifiction of (c). e, Representtive immunocytochemicl imges depicting levels in primry hippocmpl neurons (DIV14) following trnsfection with the AAV-shRNA constructs; scle r, 1μm. f, Quntifiction of (e). g, Schemtic of the experimentl timeline. Mice were 3-4 months of ge when p25 ws induced. Note tht shrna2-1 ws used s sh for ll in vivo experiments. h, Representtive pictures of mcherry showing comprle infection rtes in hippocmpl re CA1 of control mice injected with scrmled shrna (, scr), mice injected with scrmled shrna (, scr) nd mice injected with the shrna ginst (, sh); scle r, 1μm. p 1; p 1; vlues re men ± s.e.m. 1

11 , scr, scr GFP HDAC1 DAPI MERGE, scr, sh 15, scr, sh HDAC1 level (men grey vlue) 1 5 c, scr GFP HDAC3 DAPI MERGE d 15, scr, sh HDAC3 level (men grey vlue) 1 5 Supplementry Figure 11. AAV-shRNA medited reduction of does not lter levels of HDAC1 nd HDAC3., c, Representtive immunohistochemicl imges depicting hippocmpl () HDAC1 nd (c) HDAC3 levels in, scr,, scr, nd, sh nimls (n=4-5 sections from 4 mice ech)., d, Quntittive ssessment of (, c); scle r, 2µm; vlues re men ± s.e.m. 11

12 Neuron counts, scr, scr, sh NeuN DAPI, scr, scr, sh Numer of NeuN-positive cells Supplementry Figure 12. Reducing does not lter the course of neurodegenertion., Representtive immunohistochemicl imges depicting NeuN stining in hippocmpl re CA1 of, scr,, scr, nd, sh nimls (n=4-5 sections from 3-4 mice ech)., Quntifiction of (); scle r, 8μm; p 1; vlues re men ± s.e.m. 12

13 , scr, scr, sh LTP fepsp slope (% seline) TBS fepsp slope (% seline) c Input-Output 1, scr vs, scr, sh vs, scr fepsp slope (mv/s) Stimultion intensity Supplementry Figure 13. Synptic plsticity is restored upon reducing., Field excittory postsynptic potentil (fepsp) slopes in hippocmpl re CA1 of, scr,, scr nd, sh nimls (n=7-8 slices from 4 mice ech)., Averge slopes of fepsp during the lst 1min of recording; smple trces ove the r chrt represent fepsps t 1min efore (gry) nd 1h fter (lck) thet-urst stimultion (TBS). c, Input/output reltionship of seline synptic trnsmission. p 5; p 1; vlues re men ± s.e.m. 13

14 Wter Mze 3.3, scr, scr, sh Distnce trvelled (m) 2 1 Swim speed (m/s).2.1 Open Field c Distnce trvelled (cm) d Time spent moving (s) e Time spent rering (s) f Time spent in center (s) Supplementry Figure 14. Overll locomotion nd nxiety ehvior is not ffected y reduction.,, Distnce trvelled () nd swim speed () were comprle etween, scr,, scr nd, sh in the wter mze tsk. c, d, Distnce trvelled (c) nd time spent moving (d) were comprle etween, scr nd, sh in n open field test. Note tht oth groups of nimls were hyperctive s compred to, scr nimls. e, f, Time spent rering (e) nd time spent in center (f) were comprle etween, scr,, scr, nd, sh mice during n open field test. p 1; vlues re men ± s.e.m. 14

15 c H2O Protein level (fold chnge of Aβ42-1) Protein level (fold chnge of Aβ42-1) H2O2 pgr1 GR1 β-actin Protein levels (fold chnge H2O) 3. H2O2 Aβ42-1 Aβ pgr1 Aβ42-1 Aβ1-42 H2O pgr1/gr d Aβ42-1 Aβ1-42 pgr1 GR1 β-actin Protein levels (fold chnge Aβ42-1) Aβ h pgr1 GR1 β-actin Aβ h pgr1 GR1 β-actin Aβ42-1 Aβ1-42 pgr1/gr e pgr1 inding to Hdc2 promoter pgr1 inding (fold chnge of h) Aβ time (h) Supplementry Figure 15. Timecourse of pgr1 nd induction following neurotoxic insults.,, Representtive imges (left) of western lot nlyses of pgr1, GR1, nd levels of primry hippocmpl neurons (DIV14-17) () 8h fter H2O2- or H2O-tretment nd () 24h fter exposure to Aβ1-42 or Aβ42-1 oligomers nd quntifiction thereof (right); pgr1 levels were normlized to totl GR1 levels. c, d, Quntifiction (c) nd representtive imges (d) of western lot nlysis of the development of pgr1 nd induction in primry hippocmpl neurons (DIV14-17) treted with A β1-42 or Aβ42-1 oligomers. pgr1 ws normlized to GR1. e, Timecourse of pgr1 inding to the GRE in the Hdc2 promoter s reveled y ChIP nlysis in primry corticl neurons (DIV1-14) treted with A β1-42. All results re from t lest three independent experiments. p 5; vlues re men ± s.e.m. 15

16 pgr1 9 9 level (men grey vlue) pgr1 level (men grey vlue) Supplementry Figure 16. nd pgr1 protein levels re concomitntly incresed in mice. Sctter plot representtion of nd pgr1 immunohistochemicl leling of neurons in hippocmpl re CA1 in versus control mice (n=5 neurons from comprle rostrocudl positions from 3 mice ech); p

17 luc GRE luc Luciferse ctivity (fold chnge of control) GR Supplementry Figure 17. GR526 potentites the trnscriptionl potentil of the GRE in the Hdc2 promoter. Luciferse ctivity of CAD cells ( 3 independent experiments) trnsfected with the proximl promoter region of Hdc2 with (ornge) or without (lue) GRE (schemtic of constructs re shown ove the grph) nd co-trnsfected with constitutively ctive form of GR, GR526 (see Methods). p 1; p 1; vlues re men ± s.e.m. 17

18 tu Hippocmpus Entorhinl Cortex c β-myloid Hippocmpus d Entorhinl Cortex BB I-II BB I-II BB I-II BB I-II BB III-IV BB III-IV BB III-IV BB III-IV BB V-VI BB V-VI BB V-VI BB V-VI Supplementry Figure 18. Tu tngles nd β-myloid ccumulte in the hippocmpus nd the entorhinl cortex s Alzheimer s disese progresses.,, Representtive imges of the cses nlyzed in this study showing the ccumultion of tu tngles (drk-rown spots) during the development of the Brk nd Brk (BB) stges of Alzheimer s disese in () hippocmpl re CA1 nd () the entorhinl cortex. c, d, Representtive imges of the cses nlyzed in this study showing the ccumultion of β-myloid (drk-rown ggregtions) during the development of the Brk nd Brk (BB) stges of the disese in (c) hippocmpl re CA1 nd (d) the entorhinl cortex; scle r, 1µm. 18

19 HDAC1 c HDAC3 BB I-II BB I-II BB I-II BB III-IV BB III-IV BB III-IV BB V-VI BB V-VI BB V-VI d protein level (fold chnge of control) e HDAC1 protein level (fold chnge of control) f HDAC3 protein level (fold chnge of control) BBI-II BBIII-IV BBV-VI Supplementry Figure 19., ut not HDAC1 or HDAC3 levels re incresed in the entorhinl cortex during the progression of Alzheimer s disese. -c, Representtive immunohistochemicl imges depicting (), () HDAC1, nd (c) HDAC3 levels in neuronl nuclei in the entorhinl cortex of ptients with Brk nd Brk (BB) stges I/II (n=4), III/IV (n=7) nd V/VI (n=7) compred to BB helthy control rins (, n=7); insets show mgnifiction of one neuron (indicted y n rrow, nucleus surrounded y white dotted circles); scle r, 1μm. d-f, Quntittive ssessment of -c, p 1; vlues re men ± s.e.m. 19

20 Supplementry Tle 1 Evidence for reduced expression of Alzheimer s disese-relted trget genes Gene Evidence Smple type Reference Arc mrna protein Humn post-mortem AD rin Amyloid-contining post-mortem humn rin regions β-myloid treted primry corticl neurons Ginserg et l., 22 Dickey et l., 25 Wng et l., 25 Bdnf mrna Humn post-mortem AD rin Philips et l., 1991; Murry et l., 1994; Colngelo et l., 22; Tn et l., 21 Cdk5 mrna Humn post-mortem AD rin Ginserg et l., 22; Ling et l., 28; Tn et l., 21 Egr1 mrna β-myloid treted humn rin pericytes Amyloid-contining post-mortem humn rin regions Homer1 GluR1 GluR2 mrna protein mrna protein mrna mrna/protein Amyloid-contining post-mortem humn rin regions β-myloid treted primry corticl neurons Humn post-mortem AD rin Humn post-mortem AD rin Humn post-mortem AD rin Glnin-negtive neurons in humn post-mortem AD rin Rensink et l., 22/4 Dickey et l., 25 Dickey et l., 25 Roselli et l., 29 Ginserg et l., 22 Wkyshi et l., 1999 Ginserg et l., 22 Counts et l., 29; Tn et l., 21 Nfl mrna Humn post-mortem AD rin McLchln et l., 1988; Clrk et l., 1989; Kittur et l., 1994; Colngelo et l., 22; Ginserg et l., 22 NR2A mrna Humn post-mortem AD rin Ginserg et l., 22; Bi nd Sze 22; Tn et l., 21 NR2B mrna Humn post-mortem AD rin Ginserg et l., 22; Bi nd Sze 22 Syp mrna Humn post-mortem AD rin Heffernn et l., 1998; Cllhn et l., 1999; Colngelo et l., 22; Ginserg et l., 22; Mufson et l., 22 Syt1 mrna Humn post-mortem AD rin Ginserg et l., 22; Mufson et l., 22; Tn et l., 21 Arc, ctivity-regulted cytoskeleton-ssocited protein; Bdnf, rin-derived neurotrophic fctor; Cdk5, cyclin-dependent kinse 5; Egr1, erly growth response protein 1; GluR, glutmte receptor; Nfl, neurofilmin; NR2, N-methyl D-sprtte receptor sutype 2; Syp, synptophysin; Syt1, synptotgmin. 2

21 Supplementry Tle 2 Primer sequences used for promoter mplifiction Promoter Forwrd (5-3 ) Reverse (5-3 ) Arc CAGCATAAATAGCCGCTGGT GAGTGTGGCAGGCTCGTC Bdnf 1 TGATCATCACTCACGACCACG CAGCCTCTCTGAGCCAGTTACG Bdnf 2 TGAGGATAGTGGTGGAGTTG TAACCTTTTCCTCCTCC Bdnf 4 GCGCGGAATTCTGATTCTGGTAAT GAGAGGGCTCCACGCTGCCTTGACG Cdk5 CGCAGCCTGTTGGACTTTGT GCGTTGCAGAGGAGGTGGTA Egr1 GTGCCCACCACTCTTGGAT CGAATCGGCCTGTATTTCAA Homer1 CTGCCTGAGTGTCGTGGAAG ATGATTTCACTCGCGCTGAC GluR1 GGAGGAGAGCAGAGGGAGAG TTCCTGCAATTCCTTGCTTG GluR2 GCGGTGCTAAAATCGAATGC ACAGAGAGGGGCAGGCAG Nfl GCCAGACGAAAGTCAGGAAG TTTTCACCAGCAGTTTGCAG NR2A TCGGCTTGGACTGATACGTG AGGATAGACTGCCCCTGCAC NR2B CCTTAGGAAGGGGACGCTTT GGCAATTAAGGGTTGGGTTC Syp CTAGCCTCCCGAATGGAATG CAGCAGCAGCATCAGCAATG Syt1 CTGAACAGGTTGAGGGCATT CCTGAGGAGAGGGGTTTAGG β-actin CCCATCGCCAAAACTCTTCA GGCCACTCGAGCCATAAAAG β-gloin TGACCAATAGTCTCGGAGTCCTG AGGCTGAAGGCCTGTCCTTT β-tuulin TCCAGGGATGAAGAATGAGG TGAGCACTGGTAGGGAGCTT Hdc2 Segment 1 TCTGGCTAGAAGCACATCCA TAGGTGTGGGCAAAAGAAGG Segment 2 CCTTCTTTTGCCCACACCTA TGGCAGACTCCTTGTATCTCC Segment 3 CCGAGTCCTGGAAATGCTTA ACCTGGTGGTCCGATATTCC Segment 4 ATATCGGACCACCAGGTAGC TTTAGCCCCTTGCTCAGAGA Segment 5 TAAGACCGAGGGGTGAACCT CCAGGGCGACAGTAGTGTTT Segment 6 CCCGTAGAAACACTACTGTCG GTAGGGCACAGAGCGGGATA Segment 7 TATCCCGCTCTGTGCCCTAC CGCCTCCTTGACTGTACGC Segment 8 CATGGCGTACAGTCAAGGAG TCTATGAGGCTTCATGGGATG Segment 9 TATTATGGCCAGGGTCATCC GACGTTAAATCTCTGCATCTGCT Arc, ctivity-regulted cytoskeleton-ssocited protein; Bdnf 1, rin-derived neurotrophic fctor, promoter region of exon I; Cdk5, cyclin-dependent kinse 5; Egr1, erly growth response protein 1; GluR1, glutmte receptor 1; Nfl, neurofilmin; NR2, N-methyl D-sprtte receptor sutype 2; Syp, synptophysin; Syt1, synptotgmin; Hdc2, histone decetylse 2; segment 1, first segment mplified in the 13p-long Hdc2 promoter region (see Fig. 3l). 21

22 Supplementry Tle 3 Primer sequences used for coding sequence mplifiction Coding region Forwrd (5-3 ) Reverse (5-3 ) Amplicon position (p from TSS) Arc GAAGTGGTGGGAGTTCAAGC CTCCTCAGCGTCCACATACA 753 Bdnf CATTCAGCACCTTGGACAGA CAGCCTACACCGCTAGGAAG 77 Cdk5 GGCTAAAAACCGGGAAACTC CCCAATACCAGCCCAGTCTA 622 Egr1 GGGAGGGTTTGTTTTGATGA TTTCAACAGCTGACGCAAAC 553 Homer1 CATAGCCAAAAGCCGGTCTA ATCATTGCCAACCTTGTTCC 645 GluR1 CACATGTAGCCGGAGTGATG CACTCAAGAGGATGGGGAAA 622 GluR2 ATTTCGGGTAGGGATGGTTC ACCATCCTTCACTGGCATTC 64 Nfl GACAGCCTGATGGACGAGAT GGCTCTTGAACCACTCTTCG 64 NR2A TGAGAATTGCTCGGTGTCTG ACCTGGCACTGTAGGAATGG 564 NR2B GGCTACGGCTACACATGGAT CCTCTTCTCGTGGGTGTTGT 582 Syp CTCCTCGGCTGAATTCTTTG CATTGGCCCTTTGTTGTTCT 36 Syt1 CGATGCTGAAACTGGACTGA GGCAGCAGGAAGACTTTGAC 363 β-actin TGTTACCAACTGGGACGACA ACCTGGGTCATCTTTTCACG 312 β-gloin CCTTTTTAGGCTGCTGGTTG AGAATAGCCAGGGGAAGGAA 2 β-tuulin GCCAAGTCACAATGGAGGTT ACAGCTTTCAACAGCCCAGT 745 Arc, ctivity-regulted cytoskeleton-ssocited protein; Bdnf, rin-derived neurotrophic fctor; p, se pirs; Cdk5, cyclin-dependent kinse 5; Egr1, erly growth response protein 1; GluR1, glutmte receptor 1; Nfl, neurofilmin; NR2, N-methyl D-sprtte receptor sutype 2; Syp, synptophysin; Syt1, synptotgmin; TSS, trnscriptionl strt site. 22

23 Supplementry Tle 4 Primer sequences used for exon mplifiction Gene Forwrd (5-3 ) Reverse (5-3 ) Arc GTTGACCGAAGTGTCCAAGC CGTAGCCGTCCAAGTTGTTC Bdnf I CTCAAAGGGAAACGTGTCTCT TCACGTGCTCAAAAGTGTCAG Bdnf II CTAGCCACCGGGGTGGTGTAA TCACGTGCTCAAAAGTGTCAG Bdnf IV TGCGAGTATTACCTCCGCCAT TCACGTGCTCAAAAGTGTCAG Cdk5 CTCATGAGATTGTGGCTCTG GACGTGGAGTACAGCTTGGC Egr1 AGCGAACAACCCTATGAGCA TCGTTTGGCTGGGATAACTC Gpdh AGAGAGGGAGGAGGGGAAATG AACAGGGAGGAGCAGAGAGCAC GluR1 GTGGTGGTGGACTGTGAATC TTGGCGAGGATGTAGTGGTA GluR2 TGTGTTTGTGAGGACTACGGCA GGATTCTTTGCCACCTTCATTC Hdc2 GGGACAGGCTTGGTTGTTTC GAGCATCAGCAATGGCAAGT Homer1 AGCAGAAGGAAGGCTTGACT CACGGTACGGCCAATAACTA Nfl AGCTGGGTGATGCTTACGAC AGCTGCACTTGAGCCTTCTC NR2A TGCAAGTTACACAGCCAACC ATCGGAAAGGCGGAGAATAG NR2B CCCAGATCCTCGATTTCATT GCCAAACTGGAAGAACATGG Syp GCCACGGACCCAGAGAACAT GGAAGCCAAACACCACTGAG Syt1 CATCGACCAGATCCACTTGT TCGTTTCCTACTTGGCACAC β-actin GGGAAATCGTGCGTGACATT CGGATGTCAACGTCACACTT β-gloin AGCTGCATGTGGATCCTGAGA GATAGGCAGCCTGCACTGGT β-tuulin TAGTGGAGAACACAGACGAGA CTGCTGTTCTTACTCTGGATG Arc, ctivity-regulted cytoskeleton-ssocited protein; Bdnf I, rin-derived neurotrophic fctor exon I; Cdk5, cyclin-dependent kinse 5; Egr1, erly growth response protein 1; GAPDH, glycerldehyde-3- phosphte dehydrogense; GluR, glutmte receptor; Hdc2, histone decetylse 2; Nfl, neurofilmin; NR2, N-methyl D-sprtte receptor sutype 2; Syp, synptophysin, Syt1, synptotgmin. 23

24 Supplementry Tle 5 Cse detils Smple ID Dignosis Sex Age (yers) Postmortem Intervl (hours) BM1 BB m BM9 BB f BM11 BB f BM16 BB f BM17 BB f MADRC912 BBIII/IV f 13 5 MADRC131 BBI/II m MADRC1314 BB m MADRC1315 BBV/VI f 64 9 MADRC1323 BBIII/IV m MADRC1325 BBV/VI f 8 3 MADRC136 BBV/VI f MADRC1368 BBIII/IV f MADRC1377 BBV/VI m MADRC141 BBIII/IV m 9 24 MADRC1424 BBV/VI m MADRC1433 BBIII/IV m MADRC1434 BBV/VI f 89 6 MADRC1454 BBI/II m 85 9 MADRC1476 BBV/VI f MADRC1477 BBI/II f MADRC1483 BBV/VI f 9 3 MADRC1492 BB f 87 4 MADRC151 BBIII/IV m MADRC1522 BBI/II f MADRC1531 BBIII/IV f MADRC1564 BBV/VI m 9 12 MADRC1576 BBI/II m 88 n.d. BB, Brk nd Brk stge; BB, cses with no neurofirillry tngles nd thus considered helthy controls; BM, Boston Medicl Center; MADRC, Msschusetts Alzheimer Disese Reserch Center t Msschusetts Generl Hospitl; n.d., not defined. 24

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION . Norml Physiologicl Conditions. SIRT1 Loss-of-Function S1. Model for the role of SIRT1 in the regultion of memory nd plsticity. () Our findings suggest tht SIRT1 normlly functions in coopertion with YY1,