doi:1.1/nture1 NF-κB/RE-GFP TNF-α pdna CA CA + DN d e NF-κB/RE-GFP GFP+DAPI NF-κB/RE-GFP GFP+DAPI NF-κB/RE-GFP GFP+DAPI V V DAPI DAPI NF-κB RE/GFP V V Mediosl hypothlmus Thlmus Cortex Suppl. Figure 1. NF-κB/RE-GFP reporter nd ging-relted rin NF-κB tivtion. &. HEK9 ells were infeted with lentivirl NF-κB reporter t low dose of DNA, treted with TNF-α (1 ng/ml) vs. vehile () (), or o-trnsfeted with CA, DN plsmid vs. ontrol plsmid pdna (), nd exmined for the indution of GFP (&). Cells in the slides were reveled y DAPI nuler stining (lue). Br = 5 µm. e. ( months) vs. old ( months) C57BL/ mie were injeted with NF-κB/RE-GFP lentivirus in the MBH (), thlmus (d), or ortex region (e). Following (~-week) omplete reovery, rin setions ross the MBH, the thlmus, nd the ortex were mde nd exmined for GFP. The entire popultions of ells were reveled y DAPI nuler stining (lue). Br = 1 µm. WWW.NATURE.COM/NATURE 1
SUPPLEMENTARY INFORMATION GFP NeuN Merge MBH MBH MBH V MBH MBH MBH p-rela RelA β-tin p-rela RelA β-tin GFP GFAP Loomotor distne (m) 9 Open Field Test. Loomotor speed (m/s) Merge..1 months post injetion Suppl. Figure. NF-κB mnipultion in the mediosl hypothlmus of mie. MBH IKKβ, MBH, nd MBH mie were generted s desried in Fig... Su res of MBH imges showing the o-loliztion of GFP with neuronl mrker NeuN ut not stroyte mrker GFAP. Br = 5 µm.. MBH smples were disseted from mie nd nlyzed vi Western lots for the levels of phosphorylted RelA (p-rela) nd totl protein levels of RelA. Blots for β-tin were used s n internl ontrol.. Mie presented in Fig. were first exmined with open field test to ssess their tehnil suitility prior to T-mze test. Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
MWM proe trils: regionl time (s) f Musle fier re (µm, X1) Visul pltform test: swimming speed (m/s) 1 1 1 Trget g MWM trining: lteny (s) Off-trget Derml thikness (mm) d MWM proe trils: lteny (s)...1. D1 D D D D5 e 1 h Bone mss: BV / TV Musle endurne (min)...1. 1 Suppl. Figure. Aging retrdtion y hypothlmi NF-κB inhiition in femles. Femle C57BL/ mie (how-fed, ~1-month-old) reeived MBH injetions of lentivirl DN (IκBα) vs. ontrol (), s desried in Fig.. At ~ months post gene delivery, following ssessment of tehnil qulifition vi swimming speed test (), mie were exmined with Morris Wter Mze (MWM) showing the performne during the trining session () nd proe trils (&d). Susequently, mie were ssessed for musle endurne (e), musle fier sizes (f), skin thikness (g), nd one mss (h). P <.5, P <.1, : n = ( d), (e), (f&g), nd 5 (h); : n = ( e, h), nd (f&g). Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION NF-κB/RE-GFP I-1 Merge+DAPI Middle 1 GFP + I-1 + (% I-1 + ) 5 Y M O Suppl. Figure. Role of hypothlmi mirogli in ging. (Y), middle-old (M), nd old (O) C57BL/ mie reeived MBH injetions of lentivirl NF-κB/RE-GFP, s desried in Fig. 1&d, nd following ~-week reovery, rin setions were mde nd nlyzed for GFP nd I-1 immunostining. Br = 5 µm. Br grph shows perentges of ells o-expressing NFκB/RE-GFP nd I-1 (GFP + /I-1 + ) mong I-1-expressing ells (I-1 + ) in the MBH. P <.1; n = mie per group. Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
I-1 Tnf mrna (u) 1 CD11 1 Cell numer (%) 75 5 5 I-1 Cre + NeuN + Cre + I-1 + Cre TNF-α Merge CD11 Y M O TNF-α Merge Middle-old (1 month post gene delivery) d e ( months post gene delivery) f distne (m) CD11 speed (m/s) 1 CD11 Swimming speed (m/s) 9 CD11 Suppl. Figure 5. Aging retrdtion y IKKβ knokout in hypothlmi mirogli.. Hypothlmi smples otined from young, middle-old nd old mie were nlyzed for Tnf mrna levels (u: ritrry unit). f. Middle-old Ikk lox/lox mie reeived MBH injetions of lentivirl CD11 promoter-ontrolled Cre (CD11-Cre). Sme mie injeted with lentiviruses lking Cre were used s ontrols (). : Upper pnel shows shemti of lentivirl CD11- Cre. Lower pnel shows perentges of ells o-expressing Cre nd I-1 (Cre + /I-1 + ) mong I-1-expressed ells (I-1 + ) in the MBH, ording to Cre nd I-1 o-immunostining. For omprison, neuronl mrker NeuN ws exmined showing tht ells immuoretive to oth Cre nd NeuN (Cre + /NeuN + ) were sent.. MBH setions were prepred from mie t 1 month vs. months post gene delivery for I-1 nd TNF-α immunostining. Br = 5 µm. Cell ounting WWW.NATURE.COM/NATURE 5
Brin weight (g) SUPPLEMENTARY INFORMATION dt re shown in Fig. d. d f: Mie were mintined until n old ge nd sujeted to open field test (d&e) nd swimming speed test (f) efore Morris Wter Mze (MWM) test in Fig. e g. P <.1, P <.1; n = mie per group (&), nd n = in nd 5 in CD11 (d f). Error rs reflet men ± SEM. N/Ikk lox/lox.. IKKβ N/Ikk lox/lox. N/Ikk lox/lox N/Ikk lox/lox β-tin Suppl. Figure. Norml rin development of N/Ikk lox/lox mie. N/Ikk lox/lox mie nd littermte (Ikk lox/lox kground) were mintined on norml how sine wening, nd mie t n dult ge were nlyzed.. Gross view of rins. Lower pnel shows Western lot verifition of redued IKKβ in the hypothlmus of N/Ikk lox/lox mie ompred to.. Brins isolted from N/Ikk lox/lox mie nd were weighed.. Coronl setions of rins from old N/Ikk lox/lox mie nd were exmined using Nissl stining. n = mie per group (), error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
MWM trining: lteny (s) MWM proe trils: regionl time (s) d 1 1 Musle endurne (min) Ikk l/l Ikk l/l NS D1 D D D D5 5 1 NS Trget e. Ikk l/l Off-trget Ikk l/l Ikk l/l N/Ikkl /l D1 D D D D5 Musle fier size (µm X1) 1 Ikk l/l Ikk l/l Skin derml lyer (mm)... Ikk l/l Ikk l/l Suppl. Figure 7. Suitility of ontrol mie in ging study. Three types of ontrol mie inluding Ikk lox/lox mie (Ikk l/l ), Nestin-Cre mie (), nd mie were ompred with N/Ikk lox/lox mie ( ) t young (~ months) vs. old (~ months) ge for musle endurne (), ognition vi Morris Wter Mze (MWM) (&), nd musle fier sizes (d) nd skin thikness (e). Performne of mie in MWM inluded dt during trining session () nd susequent proe trils whih show time in trget nd representtive off-trget qudrnt (). P <.5, P <.1, NS, non-signifint; n = (young or ), (young Ikk l/l or N- Cre), (old or ), nd (old Ikk l/l or ) per group in pnels, nd n = per group in pnels d&e. Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE 7
SUPPLEMENTARY INFORMATION loomotor distne (m) loomotor distne (m) g d MWM trining: lteny (s) 1 1 5 15 1 5 N/Ikk lox/lox N/Ikk lox/lox N/Ikk lox/lox, young loomotor speed (m/s) e loomotor speed (m/s)... 1...5. 1.5., young D1 D D D D5 h N/Ikk lox/lox N/Ikk lox/lox MWM proe trils: regionl time (s) 1 Visul pltform test: swimming speed (m/s) f Visul pltform test: swimming speed (m/s) 1, young N/Ikk lox/lox N/Ikk lox/lox NW NE SW SE N/Ikk lox/lox, young Suppl. Figure. Additionl results from ehviorl tests of N/Ikk lox/lox mie. N/Ikk lox/lox mie ( ) nd littermte (Ikk lox/lox kground) were mintined on norml ow sine wening. At ges of 1 months ( ) vs. months (d f) ges, mie were sujeted to open field test (,, d, e), visul pltform test (&f), followed y Morris Wter Mze (MWM) test. Dt in g&h show MWM results for young mie inlude trining session (g) nd proe trils (h) whih revel time distriution in trget northwest qudrnt (NW) vs. off-trget northest (NE), southwest (SW) nd southest (SE) qudrnts. Dt for old mie re shown in Figure. : n = 1 ( ) nd 1 (d h); Ikk lox/lox : n = 1 ( ) nd 1 (d h). Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
15 1 5 d e Trget Off-trget f MWM trining: lteny (s) D1 D D D D5 MWM proe trils: regionl time (s) 1 g h i j k 1..... N/Ikkl/l Musle endurne (min) 1 loomotor distne (m) Musle fier (µm X1) loomotor speed (m/s) Skin thikness (mm).1. Bone mss: BV / TV.1. Visul pltform test: swimming speed (m/s) MWM proe trils: lteny (s) Tendon reking time (min) 1 1 Suppl. Figure 9. Aging retrdtion in femle N/Ikk lox/lox mie. Femle N/Ikk lox/lox mie ( ) nd littermte (Ikk lox/lox kground) were mintined on norml how sine wening. At n old ge, fter the ssessment of tehnil qulifition vi open field (&) nd swimming speed test (), mie were ssessed for ognition vi Morris Wter Mze (MWM) (d f), musle endurne (g), musle fiers (h), skin (i), one mss (j), nd til tendon reking time (k). MWM results show performne during trining session (d), nd time distriution in trget vs. representtive off-trget qudrnt (e) nd lteny (f) during susequent proe trils. P <.5, P <.1; : n = 11 (, g), (d f), (h&i), nd (j&k); : n = 9 ( g), (h&i), (j), nd (k). Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE 9
SUPPLEMENTARY INFORMATION.5 Gnrh1 mrna (u). Serum testosterone (ng/ml). 1.5 1..5 Referene 1.5 1..5 Referene Con Gnrh1 promoter (hnge reltive to ontrol) d Serum estrdiol (pg/ml) 1... 75 5 5 Control Referene Con Control Clphostin-C Suppl. Figure 1. Additionl nlyses of nd sex hormones.. Gnrh1 mrna levels (u: ritrry unit) were determined for hypothlmi from MBH, MBH nd MBH mie. Smples from young mie were inluded to provide norml referene.. GT1-7 ells were trnsfeted with Gnrh1 promoter reporter, o-trnsfeted with CA or ontrol plsmid, treted with PKC ntgonist Clphostin-C vs. vehile (), nd mesured for Gnrh1 promoter tivities. The levels in eh of two Control groups were normlized s 1, nd levels of group were lulted reltively to the orresponding ontrols. &d. Serum testosterone () nd estrdiol (d) in mle () nd femle (d) MBH, MBH, nd MBH mie. Smples from young mie were inluded s norml referenes. P <.5, P <.1, P <.1; Crtl: n = (), (), (&d); : n = (), (), (), nd 7 (d); : n = (), (), (), nd (d); referene: n = (), (), 5 (), nd (d). Error rs reflet men ± SEM. 1 WWW.NATURE.COM/NATURE
MWM proe trils: regionl time (s) 1 MWM trining: lteny (s) 5 1 Visul pltform test: swimming speed (m/s),, D1 D D D Trget,, Off-trget D5 Suppl. Figure 11. Additionl informtion on nti-ging effets of tretment. Mle MBH mie () nd MBH mie () reeived dily vs. vehile () injetions, s desried in Fig. f i. Dt show swimming speed (), lteny in trining session of Morris Wter Mze (MWM) test (), nd time in trget vs. off-trget regions in MWM proe trils (). P <.5, P <.1; n = (, ), 7 (, ), 7 (, ), nd (, ). Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE 11
SUPPLEMENTARY INFORMATION Trget Off-trget d e 5 MWM proe trils: regionl time (s) Visul pltform test: swimming speed (m/s) 15 1 5 1 MWM trining: lteny (s) MWM proe trils: lteny (s) D1 D D D D5 1,,,, f g h i 1.. 1.. Musle fier (µm, X1) Derml thikness (mm).1. Bone mss: BV / TV..1. Musle endurne (min) 1 Tendon reking (min) Suppl. Figure. Anti-ging effets of tretment in femles. Femle MBH mie () t n old ge were dily injeted with vs. vehile () suutneously for weeks, nd ssessed for swimming speed (), ognition vi Morris Wter Mze (MWM) ( d), musle endurne (e), musle fiers (f), skin (g), one mss (h), nd til tendon reking time (i). MWM results show lteny during trining session (), nd time in trget vs. off-trget regions () nd lteny (d) in proe trils. P <.5, P <.1; : n = ( d), (e), (f h), nd 5 (i); : n = ( d), (e), (f h), nd (i). Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE
Visul pltform test: swimming speed (m/s) 9 Trining: lteny (s) 1,, D1 D D D D5 Trget Off-trget d e 5 MWM proe tril: regionl time (s) 15 1 5,, MWM proe trils: lteny (s) 1 Musle endurne (min) 1 Suppl. Figure 1. Anti-ging effets y NF-κB inhiition nd. Mle MBH mie () t n old ge were dily injeted with vs. vehile () suutneously for 5 weeks, nd ssessed for swimming speed (), ognition vi Morris Wter Mze (MWM) ( d), nd musle endurne (e). MWM results show lteny during trining session (), nd time in trget vs. off-trget regions () nd lteny (d) in proe trils. P <.5, n = () nd () per group. Error rs reflet men ± SEM. WWW.NATURE.COM/NATURE 1