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1 . 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, nd potentilly dditionl proteins, to restrict the expression of mir-134 nd tht, () upon SIRT1 loss-offunction, higher levels of mir-134 negtively regulte synptic plsticity vi the trnsltionl lock of key plsticity proteins such s CREB. 1
2 5 Tone.2 Percentge of freezing CTR time/tot time c d Escpe Ltency (s) speed (m/s) Trils S2. SIRT1 loss-of-function impirs lerning nd memory formtion. () Tonedependent freezing ehvior is reduced in SIRT1Δ mice compred with control () mice (SIRT1Δ, n = 13;, n = 15, p <.5). () Locomotor ctivity ws not chnged in SIRT1Δ mice in comprison with controls for time spent in the center (p=.6157). (c) SIRT1Δ mice exhiit norml locomotor ctivity during the wter mze test. Escpe ltencies of control nd SIRT1Δ mice in the visile pltform wter mze test. (d) The velocities of control nd SIRT1Δ mice during the proe test were similr. 2
3 SIRT1Δ fepsp Amplitude (% mx) PPF rtio µv ms SIRT1Δ Stimulus (mv) S3. Bsl signling properties re not ltered in SIRT1Δ CA1 neurons. () The mplitude of the evoked fepsp in SIRT1Δ mice ws similr to tht in control mice. () No differences etween control nd SIRT1Δ mice were oserved in pired-pulse fcilittion. 3
4 2. SIRT1Δ ChIP with CREB fter FC SIRT1Δ fter FC Reltive q-pcr signl # # BDNF1 BDNF2 BDNF4 BS FC BS FC nti-pcreb nti-creb nti-gapdh S4. SIRT1 loss-of-function reduces the ssocition of CREB with BDNF in the hippocmpus. () Chromtin immunoprecipittion (ChIP) with n nti-creb ntiody followed y semi-quntittive PCR demonstrted strong ssocition of CREB with BDNF 1, 2 nd 4 genes in floxed controls, ut not in SIRT1Δ, hippocmpl smples (n=3). Moreover, enrichment of BDNF1 nd 4 ws enhnced fter 3 min fer conditioning stimultion in control mice, ut not in SIRT1Δ mice. () The level of phosphorylted CREB ws enhnced oth in control nd SIRT1Δ mice t 45 min fter fer conditioning trining. p <.5, # = not significnt. 4
5 S5. The expression of numer of rin-enriched micrornas is ltered in SIRT1Δ mice. A het mp digrm of the expression profile of highly enriched micrornas in SIRT1Δ hippocmpi nd littermte control hippocmpi ws generted from microrna microrry (Exqion, Inc. mircury LNA Arry version 11.). Ech row represents mirna nd the column represents the smple. The color scle illustrtes the reltive expression levels of mirna cross smples: red color represents n expression level lower thn the men, green color represents expression level ove thn men. Among the micrornas differing in expression etween the smples, mir-34-5p, mir-126-5p, mir-144, mir-126-3p, mir-451, mir-34c, mir-153, mir-19, mir-32, mir-78, mir-23, mir-16, mir-21, mir-134 mir31 nd mir22 were upregulted y more thn 25% in SIRT1Δ hippocmpi compred with control, while mir-187, mir-467, mir-221 nd mir-872 were downregulted y more thn 25%. 5
6 Reltive Luciferse Unit Scr-miR mir-134 NS mir-34c NS mir-34-5p S6. The CREB 3 UTR contins three puttive mirna-134 inding sites nd overexpression of mir-34c or mir-34-5p did not ffect CREB ctivity in cultured CAD cells. () Sequence lignment of mir-134 inding sites (predicted using the Mirnd lgorithm) in the CREB 3 UTR. () CREB ctivity ws not ltered fter trnsfection of mir34c (p=.32, n=3) or mir-34-5p (p=.62, n=3), two other micrornas found to e upregulted in SIRT1Δ mice. p <.1. 6
7 Reltive q-pcr mir-134 Level Lv-Scr-miR Lv-miR-134 fepsp Amplitude (% mx) Lv-Scr-miR Lv-miR Stimulus (mv) c Percentge Freezing Tone d Response to ES (cm/s) Lv-Scr-miR Lv-miR-134 Lv-Scr-miR Lv-miR-134 Trining ES S7. Overexpression of mir-134 in the hippocmpus does not ffect tone-dependent fer conditioning or locomotion in mice. () Rel-time PCR demonstrted upregulted mir weeks fter lentivirus expressing mir-134 (Lv-miR-134), or control virus (Lv-ScrmiR), ws injected into the dorsl hippocmpus. () The overexpression of mir-134 in CA1 hd no effect on evoked fepsp mplitude. (c) Six weeks fter receiving hippocmpl injections of Lv-miR-134, mice did not exhiit impired freezing ehvior during the tone memory test compred with Lv-Scr-miR-injected controls (Lv-miR-134, n = 14; Lv-Scr-miR, n = 12, p=.657, Student s t-test). Freezing ctivity is displyed s verge ± SEM. (d) Velocity of oth groups during the trining nd the electric foot shock (I=.8 ma). p <.1. 7
8 LNA-miR-134 DAPI merge.9 Reltive mir-134 signl LNA-scr-miR + LNA-miR-134 c 5 Tone Percentge Freezing LNA-Scr + LNA-Scr + LNA-miR-134 d Response to ES (cm/s) LNA-Scr + LNA-Scr + LNA-miR-134 Trining ES S8. Injection of LNA-miR-134 downregultes mir-134 levels in the hippocmpus of SIRT1Δ mice. () Representtive imges showing the expression of fluorescently-leled scrmled microrna in re CA1 of SIRT1Δ mice 2 dys fter injection. () Rel-time PCR demonstrted decresed mir-134 levels in the SIRT1Δ hippocmpus 2-3 dys fter LNAmiR-134 injection compred to LNA-scr-miR injection. p <.1, Student s t-test. (c) The impired tone-dependent memory in the SIRT1Δ mice ws not rescued y LNA-miR- 134 injection into hippocmpus. p <.5. (d) The response to ES of ll groups during the trining nd the electric foot shock (I=.8 ma). 8
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