TERT Inhibition Leads to Reduction of IL6 Expression Induced by Palmitic

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This article isThis article by protected copyright. All rightsreserved. 10.1111/jne.12768 doi: differences to lead between the of thisversion and Version Rec copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot 4096).Email:Phone: +57 1 3208320(ext Ciencias, de Facultad Bioquímica, * Universidad Medicine, Antonio Nariño,Bogotá, Colombia b BogotáJaveriana, D.C.,Colombia a Running title: Article :Original Article type 0000 ID (Orcid : E.BARRETO GEORGE PROFESSOR YeimyGonza

Laboratory of Neuropsychiatric Genetics, Biomedical Sciences Research Group, School of School Group, Research Sciences Biomedical Genetics, Neuropsychiatric of Laboratory

Accepted Universidad Pontificia Ciencias, de Facultad Bioquímica, y Nutrición de Departamento Article TERT Correspondence: George E. Barreto, M.Sc., Ph.D. Departamento de Nutrición y y Nutrición de Departamento Ph.D. M.Sc., Barreto, E. George Correspondence: interferes with interferes inhibition leadstoreduction of inhibition lez TERT in andtibolone astrocytes - Giraldo

the a ,

Angie V.Garzón protective oftibolone effects

Pontificia Universidad Javeriana, Bogotá D.C., Colombia. Colombia. D.C., Bogotá Javeriana, Universidad Pontificia

[email protected] IL6 expression inducedIL6 expression by - Benite -

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Diego A.Forero -

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George E.Barreto cell model model

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This article isThis article by protected copyright. All rightsr expression gene of regulation and protection (mtDNA) DNA functions function a is Telomerase Introduction i Key conclude that telomerase couldplay a role in dual cells. these and PA by inflammation in involved was about findings novel provide i tibolone Moreover, with interfered it and expression IL6 reduced with combination showed results tibolone with treated under evaluated were acid study especially evidence growing non t that shown been has it Although Abstract Acceptednterleukin 6. Article - words: aoia fntos n ernl cells neuronal in functions canonical (PA) was to evaluate the role of role the evaluate to was being ,

such as reduction of reactive oxygen species (ROS) species oxygen reactive of reduction as such and tibolone. Cell death, reactive oxygen species production and IL6 expression IL6 and production species oxygen reactive death, Cell tibolone. and on

Astrocytes

the the

the maintenance of telomere length in dividing cells dividing in length telomere of maintenance the that that regulation of pro of regulation ribonucleoprotei iooe and tibolone showing ncreased eoeae protein telomerase

and ; eoeae i telomerase p almitic acid; almitic P A

707 , ht eoeae ly a important an plays telomerase that the

using that - telomerase regulation of telomerase by telomerase of regulation Tyr phosphorylation in phosphorylation Tyr ivle i svrl ellr processes cellular several in involved n - lmrs hs erpoetv effects neuroprotective has elomerase hbto with nhibition inflammatory cytokine gene expression. The aim The expression. gene cytokine inflammatory

its

looer, lw yoer, E cytometry, flow fluorometry, t

ibolone ciiy a afce b P. eoeae inhibition Telomerase PA. by affected was activity was , t role its in

in an astrocyt an in the nrae b P atr 36 after PA by increased rtcie fet b tbln. hrfr, we Therefore, tibolone. by effects protective ; eserved. t elomerase activity elomerase protective effects protective the the o n glial cells cells glial n BIBR1532 compound in T98G cells cells T98G in compound BIBR1532

PA e cell e

- treated

PA and cell death cell and have

model

s tl ukon Tee is There unknown. still is

. and tibolone. Telomerase tibolone. and ; of tibolone of

How TERT role on inflammation, inflammation, on role cells. cells. also LISA treated

ever , hours en reported been ; mainly

In this study, we study, this In t wt i with , n qPCR. and elomere length elomere , , mitochondrial ,

non on cell death. cell on aoe rel="tag">r in or alone , with palmitic with - u to due canonical ts of this this of main

Our (

1 its ) ; .

This article isThis article by protected copyright. All rightsr asAD) (such the associated reduction length overweight and inflammation ( disease Alzheimer’s with associated is length telomere of ( consequences associated its and instability genome sequences repetitive are Telomeres ( mechanism translocated from t 6 interleukin (N B kappa factor nuclear LPS different respectively 3q26.2, and 5p15.33 in located are subunit composed is Telomerase elomerase 1 10

Accepted3 Article ) ) above factors can interact can factors above . . )

the the ( eoee hreig s rgee b t by triggered is shortening Telomere 4

(TERT) )

molecules nucleus hog atvto o svrl rncito factor transcription several of activation through

with rti i rgltd by regulated is protein

( from

8 . aog te factors other among , ( )

IL ,

, as well as well as , a b - and

F . 6 eing found eing the cell nucleus cell the l n women in ) onger ,

( r xml, ne cniin o o of conditions under example, or such as hormones as such a 6

) telomerase RNA component (TERC). component RNA telomerase , tumor necrosis factor ( factor necrosis tumor

telomere length length telomere of F a decline in decline a -

in kB) ( w subunits two to 16 mitochondria mitochondria

increas - to factor factor 18

that post ( the the ) 15 .

( ) cap the ends of ends the cap neetnl, long Interestingly, 3 - Moreover . cytoplasm by means of a phosphorylation a of means by cytoplasm e rnltoa modification translational estrogen to increase increase to ) (

the the 19 and inflammatory stimuli inflammatory and the : )

( risk of develop of risk and 9 TNF ) e increase he eserved. (

s, 2 and t

) lmrs rvre transcri reverse elomerase

reduction of AD risk AD of reduction . , have ) senescence or apoptosis or senescence

Telomerase expression is modulated by modulated is expression Telomerase eea clinical several the the and possibly in the the in possibly iaie stress xidative the the

also expression interleukin 1 beta ( beta 1 interleukin

- em hormon term (AD) chromosome s

Human ing in been associated with telomere telomere with associated been ( 5

)

TR i required is TERT . oxidative stress stress oxidative neurodegenerative diseases diseases neurodegenerative ( 12 i odr to order in s

(e.g ) of cytokines, cytokines, of TERT atr sc a o as such factors ,

endoplasmic reticulum reticulum endoplasmic

and other pathologies other and t elomerase protein protein elomerase ,

teay (HT) therapy e ( l in order to order in ipopolysaccharide 20 ) and

) IL ( . Therefore, . ptase 11 -

1B TERC ) e exported be . -

Reduction dependent ) ( including

14 catalytic catalytic

( prevent prevent 7 by the the by

) ) besity genes .

The The and all is is is ,

This article isThis article by protected copyright. All rightsr Acceptedwe Therefore, (such cells in increased is TERT that Considering mechanisms inastrocytesbrain. inthe deprivation glucose showe scar glial format in involved be could TERT that suggesting expression, (GFAP) protein acidic fibrillary glial in increase an with correlated was expression TERT that showed collaborators in increased astrocytes, other among excitotoxicity, stress, oxidative against neurons protect to injuries different in upregulated is subunit, TERT especially protein, ArticleTelomerase to ability activate estrogen receptors America Latin synthetic Otherwise, and acid death cell and stress oxidative interest particular of acid fatty of concentrations high have people Obese s TERC

on telomere on d that TERT overexpression reduced astrocyte proliferation in a model of hypoxia and and hypoxia of model a in proliferation astrocyte reduced overexpression TERT that d ion and that it could contribute to astrocyte activationastrocyte to contributecould it that and ion as

steroid used as used steroid el death cell

tibolone h rl of role the

ee expression gene the canonical and non and canonical the

oes f shmc brain ischemic of models n countries n evaluated telomerase expression, protein activity and telomere length in cells cells in length telomere and activity protein expression, telomerase evaluated

length ) ( alone induced a small increase small a induced alone o ta i cud e novd n h poetv efcs f tibolone. of effects protective the in involved be could it that or , 31 ( 21 ) ET s tl unknown. still is TERT . Thus, TERT could be involved in both detrimental and protective protective and detrimental both in involved be could TERT Thus, .

is unknown. is ) hormone replacement therapy by therapy replacement hormone

. (

25 T

i strtd at ai i al t idc inflammation induce to able is acid fatty saturated his and )

hshroe a anti has hormone This . ( 23

rae wt PA with treated

) - that

canonical functions of telomerase, of functions canonical

in ( 27

We have previously previously have We in vitro vitro in ) this this .

( 29 effect models ) eserved. i order in , n sia cr injur cord spinal and oe tde hv fud ht ET is TERT that found have studies Some s was

in in serum in .

However, the effect of saturated fatty saturated of effect the However, - TERT inflammatory effects effects inflammatory

eesd by reversed observed that PA increases PA that observed

women ( o reduce to 30

, expression expression with ) . Nevertheless, another study another Nevertheless, . injurie in several several in palmitic acid (PA) acid palmitic

iooe pretreatment. tibolone its we have we s ies ( ( ermna effect detrimental 24 28

) ) ( . . ( 30 Europe 26 Tibolone is a is Tibolone hypothesized However, in in However, ) ) To and Tao .

u to due an

TERT being being (

22 and and its its ) s ,

This article isThis article by protected copyright. All rightsr in dissolved was Tibolone hours. nM 10 stim before hours 24 for performed was treatment L and red phenol without DMEM deprived were cells NHA and T98G Drug Treatments ahumidified atkept in 37 incubator °C and5%CO (hEGF) factor growth epidermal GA with mg penicillin/10 (NHA) U 10 and France) (Eurobio, Normal USA). Walkersville, (Lonza, amphotericin ng streptomycin/25 (FBS) serum bovine fetal 10% with USA) Walkersville, (Lonza, glucose high (DMEM) Medium Eagle Modified CRL (ATCC® cells T98G Cell culture and Materials methods markersastrocyte ( from derived understand death, cell measured we and compound P to exposed Accepted Article32 ) . This cell line cell This . ulated with 70 µM tibolone (Sigma, St Louis, MO, USA) and NHA cells with 20 µM and and µM with 20 cells NHA and USA) MO, Louis, (Sigma,St tibolone µM 70 with ulated

tibolone (Lonza, - 1000,

t e oe f TERT of role he

A and tibolone. Furthermore, tibolone. and A a

Nx, mM 1 Next, . ua g human Walkersville, USA) were grown were USA) Walkersville, , % r 1%

such asGFAP fibrillary acidicprotein), and (glial nestin, vimentin has been used been has ecombinant human insulin, insulin, human ecombinant

lioblastoma - 60) Mnsa,A UA wr maintained were USA) (Manassas,VA, 1690™)

PA in

n L and

9G el under cells T98G imethyl sulfoxide (DMSO), and PA and (DMSO), sulfoxide imethyl as (Sigma, St Louis, MO, USA) was added to cells for 24 24 for cells to added was USA) MO, Louis, St (Sigma, - ltmn (oz, akrvle UA. Then USA). Walkersville, (Lonza, Glutamine

a glial cell model cell glial a tumor from serum and other supplements other and serum from - glutamine ROS we ,

hc hs nra sainr paeG arrest G1 phase stationary normal a has which inhibited t inhibited production plcto of application eserved.

in 2

. Lna Wlesil, S) Cls were Cells USA). Walkersville, (Lonza, Astrocyte

ifrn stimuli different FBS in vitro in elomerase

, ,

n I6 expression IL6 and a scorbic scorbic

Basal M Basal m P. 9G el were cells T98G PA. mM 1

( 33

function - 35 . a was dissolved was ) h cid cid edium , and expresses some some expresses and ,

uman astrocyte cells astrocyte uman T98G for 24 hours using using hours 24 for

s using a chemical chemical a using olution, , ,

in supplemented supp s cl line cell a is ,

Dulbecco’s i odr to order n ,

lemented tibolone tibolone ( in 36 human human 2.5% 2.5% ) .

This article isThis article by protected copyright. All rightsr permeabilized and fixed harvested, were cells finished, were treatments the once Briefly, expressionanalyzing protein status phosphorylation and expression to performed was analysis cytometry Flow expressionProtein and analysis using flowphosphorylation cytometry Mean nm). 540 emission nm/ 485 (excitation fluorescence (a.u.)forallexperiments. ispresentedinarbitrary units DCFDA nm), 590 emission nm/ 530 (BMG Germany) Ortenberg, reader LABTECH, microplate Omega FLUOstar a using detected was Fluorescence minutes. Both respectively. hydro and superoxide quantify to used were USA) MO, Louis, (Sigma,St described used p with Staining deathandCell production Reactive OxygenSpecies analysis ( reduce to able is compound this that observed interacti by activity DMSO. in dissolved also was compound before µM 100 at added was telomerase, of inhibitor an USA), Ca, Cruz, Santa Biotechnology, Cruz final the at cells to b 40 Accepted Article (BSA) albumin serum ovine ) .

t 0 µg/ml 10 at treatment with treatment ( 37 ) Dhdotiim DE ad 2′,7′ and (DHE) Dihydroethidium . ropidium ropidium o vlae el death cell evaluate to co compounds compounds ng ncentration 1 mM PA or with tibolone with or PA mM 1 ih mtf novd n N binding DNA in involved motif a with i odide (PI) (Santa Cruz Biotechnology, Santa Cruz, Ca, USA) was USA) Ca, Cruz, Santa Biotechnology, Cruz (Santa (PI) odide that allows obtaining

and 2 mM Carnitine mM 2 and were added to cells at at cells to added were s

used

using the using , due to due ,

in PA and tibolone treatments tibolone and PA in .

el wr sand o 1 minutes 15 for stained were Cells IB52 ind BIRB1532

DH and PI parameters: following the fact that it that fact the TERT eserved. eemn cags in changes determine

and PA and results . C . -

ihooloecn ictt (DCFDA) diacetate Dichlorofluorescin gene and gene ontrols ontrols

, as previously described previously as , that are similarthat are a uces uces concentration of 10 µM for 25 25 for µM 10 of concentration

is a very efficient technique technique efficient very a is (DMSO and BSA) and (DMSO telomerase

( n niiin of inhibition an 39 )

. Moreover, it has been been has it Moreover, .

( 37

to gen peroxide gen ) t

. BIRB1532 (Santa (Santa BIRB1532 . lmrs protein elomerase protein expression expression protein

western blot ,

s previously as E (excitation (excitation E were added added were telomerase ( 38

) levels .

( This 41 for for ) . , This article isThis article by protected copyright. All rightsr USA). Ma, Waltham, Scientific, Fisher (Thermo 1X cocktail inhibitor Thermo and orthovanadate sodium mM 1 pyrophosphate, sodium mM 2.5 fluoride, sodium mM 10 contained which USA), Ma, Waltham, Scientific, Fisher by published for instructions the followed We analysisTelomerase activity expression analysis. and USA) MA, (Millipore, Software fluorescence only incubation the to corresponded control negative The scatt forward for area the against width the plotting performed was discrimination Doublet acquisition ( 488 Dylight ( ( (Tyr707) the were used dilutions and following: Antibodies USA). MA, (Millipore, cytometer fluorescence the EasyCyte quantify to BSA 2% in resuspended and again washed the out with incubation 75% using Accepted Article Scientific, Fisher Thermo

. Next, cells were washed were cells Next, . secondary antibody (Invitrogen, Carlsbad, Ca, USA) for 1 hour. Finally, cells were were cells Finally, hour. 1 for USA) Ca, Carlsbad, (Invitrogen, antibody secondary wtot h piay antibody) primary the (without An i An

levels were hro ihr Scientific, Fisher Thermo anti e

intensity hnl o 3 mnts After minutes. 30 for thanol Herbert et al al et Herbert Thermo ceeti tefursec nest niae a increase an indicates intensity fluorescence the in ncrement - the the ET ( TERT : forward and forward : .

primary polyclonal antibody (Invitrogen, Carlsbad, Ca, USA) Ca, Carlsbad, (Invitrogen, antibody polyclonal primary

was Fisher Scientific, Fisher hro ihr Scientific, Fisher Thermo calculated ( 42

again, A387, :0 ad anti and 1:100 PA538817), s ) . ide scatter plot scatter ide Total p Total

and 30 minutes of blocking was performed before before performed was blocking of minutes 30 and

the the automatically from 5000 events events 5000 from automatically n 50 cls ee curd o ec sample. each for acquired were cells 5000 and aa ee omlzd n re to order in normalized were data rotein was extracted using using extracted was rotein 55) 1:400. 35553), A108, :0 anti 1:50; PA513048), telomeric repeat amplification (TRAP) protocol protocol (TRAP) amplification repeat telomeric washing s

w eserved. ere

A146, :0 anti 1:50; PA511446), used to identify intact cells from debris. debris. from cells intact identify to used the the f cells of aaees o f for Parameters

el wt 2 BSA, 2% with cells - abt g scnay antibody, secondary IgG rabbit with the secondary antibody antibody secondary the with - Phospho the cetfc h Scientific using the Guava Suite Suite Guava the using RIPA buffer (Thermo buffer RIPA o yoer data cytometry low perform - in TERT (Ser227) (Ser227) TERT - Phospho

with rtis were Proteins TERT protein protein TERT an overnight overnight an

alt protease protease alt was carried carried was

statistical a Guava Guava a - adding adding TERT Mean Mean er. er.

This article isThis article by protected copyright. All rightsr CFX96 a on realized was PCR Time Real µl. 15 of volume total a for water, and primer each of µM 0.8 DNA, of ng 20 UK), London, (Bioline, HRM SensiMix 2× of µl 7.5 used TT CTG CGC GGT CAT AAG GAA CCG CCG GTC CCC CC CGG GCC ALBD: and TTG TCC GAA ACA TGC TGC AAA CGG GGG TC CTA (single Albumin TCC CTA TCC CTA TCC CTA TCC GTA TAG TGT TELC: TE were: used primers The ng/µL. 10 to normalized and USA) Ca, Carlsbad, (Invitrogen, fluorometer method method a following analyzed was length Telomere lengthTelomere analysis c as used was buffer (Ct) cycle threshold cycles 40 CA). Hercules, (BioRad, System s performed was incubation ng 2 primer, ACC) CTA ACC CTT ACC CTT ACC CTT CGG (GCG ACX µM 1 and primer GTT) AGA AGC TCG No SYBR® for ng/µL 2 to normalized of means by quantified ubstrate by tel by ubstrate Accepted wastakenontrol sample as Article ( s rvosy described previously as 46 LG: ACA CTA AGG TTT GGG TTT GGG TTT GGG TTT GGG TTA GTG T and T GTG TTA GGG TTT GGG TTT GGG TTT GGG TTT AGG CTA ACA LG: at at ) - . DNA was quantified with a Qubit dsDNA BR Assay and measured in a Qubit Qubit a in measured and Assay BR dsDNA Qubit a with quantified was DNA . 95°C for 15 sec and 60°C for 60 sec. Data analysis was performed based on the the on based performed was analysis Data sec. 60 for 60°C and sec 15 for 95°C ROX Master mix (Bioline, London, United Kingdom), 1 µM TS (AAT CCG CCG (AAT TS µM 1 Kingdom), United London, (Bioline, mix Master ROX omerase. Later, R Later, omerase. - copy gene) primers gene) copy a ,

negative control. Data are shown in percentage, where percentage, in shown are Data control. negative as previous studies previous as a

TRAP of BCA kit (Thermo Fisher Scientific, Waltham, Ma, USA) and and USA) Ma, Waltham, Scientific, Fisher (Thermo kit BCA at 100% a

rti ad water, and protein 0C o 3 mn n h dr t alw h etnin of extension the allow to dark the in min 30 for 30°C

The PCR p PCR The eal

( analysis. TRAP master mix consisted in 2X SensiFAST™ SensiFAST™ 2X in consisted mix master TRAP analysis. 45 ctivity -

Time PCR was realized on a CFX96 Touch Real Touch CFX96 a on realized was PCR Time ) were .

Genomic ( . : ALBU: CGG CGG CGG GCG GCG CGG GCT GCT CGG GCG GCG CGG CGG CGG ALBU: : 43

previously used protocol used previously rogram consisted consisted rogram ) . All samples were run in duplicate and RIPA and duplicate in run were samples All . eserved. DNA o a for

a etatd sn te salting the using extracted was

oa volume total ( 45 of

) one . For PCR amplification PCR For .

cycle cycle ( 44 of )

based on the qPCR the on based 5 L A µL. 25 at at the the 95°C for 2 min, 2 for 95°C CA ACA CTA C C GCC GCG GCG GCC C value for the the for value initial n - Time , -

out the the we .

This article isThis article by protected copyright. All rightsr Accepted CT comparative the method using (2 performed was analysis Data triplicate. in run were samples AC CTC CCA CAG AGA ( primer forward gene, IL6 G); GAC ATT CTT ATC GAC GAA (TCT primer r and A) ATA AAG TCA AGA AAG GCC (ATC primer forward gene, RPL27 for control program LinRegPCR using determined specificity primer the verify to out carried was analysis cycles 40 min, 2 for °C 95 Real Touch and cDNA of µl 1 Reverse), and (Forward No SYBR® RNA. ArticleQuantitative of ng 400 from cDNA generate to used were Kingdom) United London, (Bioline, M USA). and with performed was isolation RNA Total Gene expressionanalysis for value Ct the to corresponds S and read 74°C at signal telomere for value Ct the to corresponds of means by TL relative the calculating Real Touch t a qatfe b mas f aorp Tem Fse Sinii, ata, M Waltham, Scientific, Fisher (Thermo NanoDrop of means by quantified was it ,

according to previous recommendations previous to according a - - L Rvre rncits (nirgn Crsa, a UA ad lg (t 18 (dt) oligo and USA) Ca, Carlsbad, (Invitrogen, Transcriptase Reverse MLV lbumin 88°Creadat signal ΔΔCT - - -

Time System (BioRad, Hercules, CA) and Ct values were acquired and used f used and acquired were values Ct and CA) Hercules, (BioRad, System Time Time Syste Time ROX Master mix (Bioline, London, United Kingdom), 400 nM of each primer primer each of nM 400 Kingdom), United London, (Bioline, mix Master ROX C fr ee expression gene for PCR ) ( 51 ) .

m (BioRad, Hercules, CA). PCR CA). Hercules, (BioRad, m

) at

and reverse primer (CCA GAT TGG AAG CAT CCA TC). CCA CAT AAG TGG GAT (CCA primer reverse and

95°C for ( 47 10 sec, 60°C sec, 10 ) the the . analysis (

49 the the w Trizol reagent (Invitrogen, Carlsbad, C Carlsbad, (Invitrogen, reagent Trizol ) RL7 ee a ue as used was gene RPL27 . ater, in a total volume of 10 µl 10 of volume total a in ater, / ratio T/S eserved. ( 50 a promd sn 2 SensiFAST™ 2X using performed was ) for . Primer sequences were the following: the were sequences Primer . 10 sec and 72°C and sec 10

, program

s rvosy described previously as ( 48 )

n PR fiiny was efficiency PCR and consisted in: consisted

for

a 15 sec. Melting Melting sec. 15 , normalization

on a CFX96 CFX96 a on one CCA CAC CAC CCA A

cycle cycle ( , USA) USA) , 45) everse . All All

A or or at T ,

This article isThis article by protected copyright. All rightsr Accepted arepresenteddata Mean as ±SEM. A USA). Tukey’s using performed CA, Jolla, La Software, (GraphPad Windows for 6.0 ANOVA way in analyzed were samples All Statistical analysis 100µg/mL at instructions manufacturer following reconstituted was protein Recombinant stimulation. PA on performed was USA) Ma, Waltham, Scientific, Fisher (Thermo were concentrations IL6 Once Article Recombinant concentration and nm 450 at Germany) LABTECH, Ortenberg, (BMG reader microplate Omega FLUOstar a in measured was Absorbance duplicate in measured e independent three from triplicates manufacturer the following performed w supernatant of µl 50 Briefly, used. 6, interleukin quantify To IL6 secretion analysis s

and . and two and h

uman

different

IL6 way ANOVA way

n h EIA microplate ELISA the in test. A test. the protein treatment protein dilutions inmedium. weredone DMEM

determined triplicate, for three independent experiments. independent three for triplicate, IL

P - 6 Human ELISA Human 6

value < 0.05 was considered significant. In the the In significant. considered was 0.05 < value

xperiments ere

tests tests

´s

obtained from obtained , a treatment with a recombinant humanrecombinant a with treatment a , a

were

ntutos Samples instructions. tnad uv ws sd o aclt protein calculate to used was curve standard

, n=9 for each treatment each for n=9 , calculated eserved. ,

codn t mnfcue´ instructions manufacturer´s to according kit (Invitrogen, Carlsbad, Ca, USA) was was USA) Ca, Carlsbad, (Invitrogen, kit a 24

using the GraphPad Prism version Prism GraphPad the using - well plate and then the assay was was assay the then and plate well (which correspond to the the to correspond (which

T98G p s hc nlss was analysis hoc ost ) ,

and standards were standards and cells cells

Student's before 1 mM mM 1 before IL6 figures protein t , o , all , ne - . This article isThis article by protected copyright. All rightsr activity stimu PA mM 1 a following tibolone length telomere and activity of function canonical The analysis + hours, 48 at However, found ( hours were observed between hours 24 in increase treatment and hours. 48 and 36 24, 12, for treatment PA mM 1 by treatment Next, 1D (Figure in evaluated T98G stimulated with cells alone tibolone and PA plus tibolone First, study, this In in T98G cell line analysis length telomere and activity telomerase expression, protein Telomerase Results

Accepted treatment PA Article

n nlss f two of analysis an e analyzed we ewe cnrl n P ( PA and control between P

a asse by assessed was = at 48at hours (

on P 0.002 protein = )

TERT expression. protein

and

0.002 we

( (.3) 2. = (6.137) F )

(

aimed and 24 and 48 hours ( hours 48 24and and P= infcn dfeecs ee o observed not were differences significant expression expression

, after rti expression protein P

= 0.0 we 12

0.012 to evaluate the role of TERT in cells treated with tibolone and PA and tibolone with treated cells in TERT of role the evaluate to PA treatment 07 - TA protocol TRAP a only

a AOA a promd o evaluat to performed was ANOVA way and 36 hours ( and 36hours telome ). A similar result was observed observed was result similar A ).

w , respectively) , 716,

at ere observed

36 P= rase is to maintain telomere length. telomere maintain to is rase

analyzed in the current study. Cells were pretreated with with pretreated were Cells study. current the in analyzed P

hours in cells treated with PA with treated cells in hours 0. = 0.004 (Figure 1A, B and C and B 1A, (Figure lus

(Figure S1)(Figure Cells were for Cells treated followed with24 hours tibolone P using ,

tibolone a significant difference between con differencebetween significant a

P = 02 and then and

= 0.0002), 48hours( 12and ) 0.009 . ) , .

The .

n NV ts ( test ANOVA An and I n cells treated with treated cells n

lw cytometry flow eserved. ) only c (Figure 1E (Figure . ot o test hoc post nrl versus ontrol total W

versus ( versus its vehicle e observed an observed e protein was isolated and isolated was protein

)

. ) (t= . TERT protein TERT by At 36 At F (3. F ,

niae ta tee was there that indicated t 0.0118 ibolone in cells treated with PA, PA, with treated cells in t , ibolone n immunocytochemistry an

the e in comparison to comparison in interaction between interaction 68)

hours, differences were were differenceshours,

P T DMSO

= h df= , 4. =

erefore, telomerase erefore, fet f time of effect 0.0008), 24 and 36 0.0008), 24and36 +

trol and trol

A ( PA 216, PA expression was expression

16, )

, for 24 hours for 24hours

P= differences telomerase telomerase P P 0.008 = = t

ibolone 0.003

12 and 12 0.91 time and and

a ) ) n ) . . .

This article isThis article by protected copyright. All rightsr Accepted with DMEMshowedthatitdoesnot peroxide affect hydrogen ( levels co by affected not were which peroxide by produced cells T98G or tibolone µM 70 PA on effect the with that showed test uptake PI PA. and tibolone with combination protein model, telomerase current of the inhibitor in specific TERT a of BIBR1532, role the investigate To tibolone. of effects protective Articlewith by death TERT production oxygen species of Effects not was it control, to comparison in statistically different. treatment PA in lower was activity telomerase between observed alone tibolone to comparison in telomerase followed the the

a no has protective effect of tibolone against cell death induced by PA by induced cell death against tibolone ofeffect protective reduction of reduction its translocation to translocation its show by

TERT (Figure S2 (Figure - a Tukey's multiple comparisons test comparisons multiple Tukey's a treated n TERT ed - aoia functions canonical that PA and tibolone plus PA induce a reduction in its levels its in reduction a induce PA plus tibolone and PA that

100 µM 100 i

nhibition using compound BIBR1532 on cell death and reactive reactive and death cell on BIBR1532 compound using nhibition cells bln + A n tibolone and PA + ibolone

inhibition Telomere was affected length byanytreatment not the the ) .

detrimental effect detrimental Quantification of superoxide of Quantification ( ANOVA test ANOVA BIBR1532

mitochondria

- treatment with BIBR with treatment , F 57) 1. = (5.78) (F ,

such as reduction of reactive oxygen species and cell cell and species oxygen reactive of reduction as such , alone or in combination, in or alone , , F (6 F ( 52 induced induced .

98) ) ( .

P 927, Our hypothesis was that that was hypothesis Our eserved. =

= 36.02, = determined that that determined

0.04 (Figure 3C) (Figure by ( P P =

0.10 = (Figure 3B) (Figure PA or or PA ; sgiiat differen significant a );

0.04 ( P 38 = <0.0001) = , ) that that ) .

39 induce Fgr 2A (Figure . Comparison of BIBR alone BIBR of Comparison . Q TERT PA reduced the reduced PA ) it could be involved in involved be could it atfcto of uantification was , did not show any effect any show not did Figure 3Cand S3) ,

but but d

(Figure 3 (Figure

(Figure 2B (Figure a inhibition inhibition

TERT

cytotoxic effect on effect cytotoxic it used d ), id

although the the although ( not have any have not is associated is ce was also also was ce ln o in or alone P A)

activity of activity <0.0001) interfered hydrogen hydrogen ) . .

Neither Neither

the the ,

This article isThis article by protected copyright. All rightsr Acceptedcells T98G pretreatment (post treatment PA mM 1 after and hours ( ELISA by determined those than concentration tibolone, pg/mL cells n The that telomerase isinvolved the in induction ofIL6 expression byPAinT98G. with observed the to similar hours), 24 as (such increase the however, PA, and control tibolone inhibitor) Article(TERT inhibition DMSO 0.017 with test comparison multiple Tukey's with interferes inhibition TERT the investigated were inhibition telomerase under secretion protein cytokine of induction the in participates telomerase As T98G treatmentcells underacid palmitic of Expression

treated with PA with treated PA

) ext step was to determine whether whether determine to was step ext before ; these co these ; (Figure 4 (Figure Fgr S4). (Figure

on IL6 on reduced reduced PA F 44) 218. = (4.40) (F ,

in comparison in (F (5.65) = 29.00, = (5.65) (F

a stimulation with stimulation a n TR inhibitor TERT and IL

ncentrations secretion A) in comparison in 6 IL6

. gene and protein secretion under secretion protein and gene

Expression of Expression . W .

An expression gene e ( ANOVA incubated cells incubated wit P 6, = were similar to those those to similar were h

0.0006) P PA cells (data not shown). We not (data PA cells

with P

1 mM PA (pre PA mM 1

showed 0. <

< IL6

test 0.

the the

PA and control ( control and PA (BIBR1532) 0001) , 0001).

was affected by BIBR1532 alone BIBR1532 by affected was (Figure 4 (Figure in comparison with cells treated with treated cells with comparison in

- IL6 ramn) W ol osre a efc b IL6 by effect an observed only We treatment). for IL6 secretion secretion IL6 for

I this cytokine this that that with L 6 by PA could arise could PA by 6

. Co . gene expression (F (4.39) = 4.51, = (4.39) (F expression gene P a treatment with treatment a a recombinant IL6 protein at 58, 298 58, at protein IL6 recombinant a rotein TERT protein expression protein TERT - eserved. - treatment), in co in treatment), B) treatment and post and treatment determined by ELISA in cells treated with treated cells in ELISA by determined . 00 pg/mL 1000 . n addition In Differences were not observed between between observed not were Differences

erto was secretion P

is beneficial or detrimental or beneficial is

TERT expression, <0.0001)

also

. found

BIBR1532 . L ws incubated was IL6 ). inhibition and its effects on on effects its and inhibition

An with longer incubation times times incubation longer with , hwd an showed

- and treatment with 1 mM PA, PA, mM 1 with treatment we tested tested we ANOVA test test ANOVA - IL6

treatment did not affect not did treatment that that eue b BIBR1532 by reduced

improved

, gene expression and and expression gene .

high concentrationshigh in comparison in These data suggest suggest data These prior fet of effect

PA hge IL6 higher a P to the effect of effect the showed = 0. =

incubation

alone for T98G T98G for

and 004

o 24 for TERT

with

). ( that that 457 P A =

This article isThis article by protected copyright. All rightsr of phosphorylation Ser ( T w site with comparison pretreatment comparison observed effect ( from ( 707 tyrosine sites: following the of status phosphorylation and activity protein responsible sites phosphorylation several has protein Telomerase Telomerase analysis phosphorylation feedback between low at effect protective in that with ( PA by induced death against d IL6 of t=3 8 ibolone ibolone

Accepted, Article ET ee expression gene TERT - 53 227 . a treatment with treatment a 913, df=16, IL6 the the

) ere of .

id hs nlss a crid out carried was analysis This

nucleus to cytoplasm) and and cytoplasm) to nucleus (t=0. affected TERT expression expression TERT affected that PA increased increased PA that treatments

was o afce b ay treatment any by affected not not increase cell death by PA, by death cell increase not

with when

47 df=16, 1427, be o induce to able P

PA ( PA = this cytokineandthethis telomerase control control telomerase its 0.001 compared IL6 n Tyr on P location in cytoplasm or nucleus or cytoplasm in location

= conc

( ) 0.013 at 58 pg/mL 58 at (

(Figure (Figure P

( entrations and entrations - b a 50% a (by = P P 0 phosphorylation 707

0.008 = =

protein P ) with 0.001

hshrlto in phosphorylation = (Figure 0.89

0.013 5C, D

s (F erine 227 ( 227 erine . This ). oto values control , )

) )

which , using = 4.459 = ) the the )

Fgr 6 , D) C, 6 (Figure ) Fgr 4 (Figure in comparison in

) 5 but , . Fgr 4 (Figure F 23) 0.08 = (2.30) (F A

Tibolone alone inducephosphorylation of alone did not Tibolone furthermore,

, B ,

phosphorylation IL6 effect f regulates its regulates low cytometry. cytometry. low , ) eserved. a . P

The pretreatment s = could existcould D) ignal required to required ignal F 22) 25.51, = (2.24) (F

C). a improved was 0.011 Tyr ( . 0 %, 90

phosphorylation levels of the the of levels phosphorylation . In the current study, current the In . hs dt sget that suggest data These with

Moreover, Moreover, - it put it 707 )

. A post hoc analysis determined analysis hoc post A . transloca Tee aa ugs that suggest data These . a 5

DMEM , i cmaio wt DMSO with comparison in , . signal required to translocate it it translocate to required signal

s P

levels P

An ( forward

at 58 pg/mL 58 at

000) and <0.0001) 0. = o te ouain of modulation the for NV ts soe an showed test ANOVA

we found that that found we tion

y 90% by , locate

of 92 resulted in an in resulted

the possibility that that possibility the P the the ) to

(Figure

<0. the nucleus) the in it ) Tyr we

protected cells protected pn tibolone upon 0001). cytoplasm

- assessed the assessed

by 0 site 707 IL6 treatment 6 increase 40% Ser , B A, It was was It has a a has - 227 ,

, the the

its its in in is is ) a .

This article isThis article by protected copyright. All rightsr and i expression, protein and gene of regulation example, for mechanisms, t The non and canonical by apoptosis from cells protect can telomerase (e.g cells mitotic types, cell different in functions several has Telomerase Discussion =11.46,(2.15) although ( control Tyr cells, T98G to wasaffectedNHAincells phosphorylation between ( change any site human normal and cells T98G on tibolone astrocytes and PA of effect the compare to order In astrocytes T and expression Telomerase compound by modified Accepted Article 1. = 3.49) )

nteraction withother protein of the telomerase protein in protein telomerase the of lmrs activity elomerase

P tibolone versus DMSO versus tibolone t induced it

(NHA) in T98G cells in

205,

0. tibolone,

00 telomerase protein telomerase P = 0. P 1 , we focused focused we , somatic cell somatic ) 0. = .

- n increase an 001 T 0 popoyain a reduced was phosphorylation 707 ibolone 32 a ) . ehns ta cudb ivle in involved be could that mechanism ) ,

at (Figure (

its a concentration of s pretreatment ) than ) on yr

aoia function) canonical (F (2.14) = 0. = (2.14) (F f 27% of s

NHA cells. Treatment with PA for 48 hours hours 48 for PA with Treatment cells. NHA

expression -

( assessing 707 phosphorylation status analysis in normal human human normal in analysis status phosphorylation 707 54 7 and A ) in .

postmitotic n hshrlto, in phosphorylation, in

(

F (3.14) =16. (3.14) F i nt ees ti effect this reverse not did

expression in comparison in B

1391, ) 10 nM . eserved.

infcn dfeecs ee not were differences Significant

cells P

(

P = 0. = and phosphorylation phosphorylation and

= y 2 upon 22% by

with 97, ( regulated 0.001 such as such 82 with P )

its expression its <0. (Figure the )

comparison

control (Figure 0001). However, contrary However,0001). contrary neurons).

- rtcie fet of effects protective aoia functions canonical protein y severa by

(Figure 7 PA or C 8

C and D , t and D and

ibolone ibolone phosphorylation with levels being compared compared In this context, this In did not induce induce not did l 8 ad B and A ) DMSO

molecular molecular

. higher in higher observed observed ( ). + Tyr Tyr

PA - - with 707 707 this this (

1 (F (F (F (F ) ) . ,

This article isThis article by protected copyright. All rightsr cytoplasm the from export its to leads protein telomerase in residue this of Phosphorylation alone supported is This death. reducing mitochondria, to nucleus from translocation telomerase induce can tibolone a and to due actions PA by induced death re Our known cells T98G in TERT of role the evaluate effects detrimental A r treatment expression expression protein affect not was reports by affected been has activity telomerase contrast, are However, activity. reverse w Here, eduction Accepted non several has protein TERT s Article no

dditionally

sults suggest that telomerase was telomerase that suggest sults

or reports showing reports d to inhibit telomerase protein through reduction of gene and protein expression protein and gene of reduction through protein telomerase inhibit to ( e found that found e

55 y iooe pretreatment tibolone by in (Figure 1E (Figure n obnto with combination in ) increased . telomerase activity We observed that protein expression of TERT of expression protein that observed We ed ( the 8 . Notably, telomera Notably, . ) by .

regula A significant differences were not observed between control and PA and control between observed not were differences significant levels induced

lthough PA also induced an increase in Tyr in increase an induced also PA lthough . t trans its PA ( ) Previously, 24 .

a Such increas Such

tion of several of tion ) in part in induced reduction in reduction

( and the the and 56 by location ) . .

Nevertheless

by PA

A nrae Tyr increased PA i a reduction reduction a t has been demonstrated that t that demonstrated been has t protein i sie that spite in , se activity can be can activity se

our analysis of phosphorylation. We found that tibolone tibolone that found We phosphorylation. of analysis our - e n el death cell on aoia functions canonical to telomerase activity telomerase could arise as a compensatory mechanism compensatory a as arise could genes

mitochondria involved in involved using level a 2 hus fe P stimulation PA after hours 24 at , , such as BAX/BCL2 as such , in

s a chemical compound chemical a

eserved. w telomerase activity telomerase . el tetd with treated cells ere

T - c modulated aking aking 707 phosphorylation phosphorylation 707 the the

is found ( - 59

nauae fty acids fatty unsaturated

, caused protection by protection ) . its

this hrfr, we Therefore, after increased increased

increase -

elomerase 707 phosphorylation, TERT TERT phosphorylation, 707 independently of independently no account into by

24 hours of PA treatment PA of hours 24 ( , but this effect was not was effect this but , a 57

iooe a a had tibolone saturated fatty acid. In acid. fatty saturated (BIBR1532) )

after 36 h 36 after

tibolone could could and

has ant has hypothesize

n 9G cells T98G in Caspase

we , the nucleus to to nucleus the attenuate ,

in previous previous in gis cell against TERT ours i its due to due - , apoptotic aimed which is which

gene or gene the the .

higher higher of PA of T

( ( gene gene

here 58 40 that cell cell

the the the t ) ) o , . .

This article isThis article by protected copyright. All rightsr Accepted mightacid bedifferentbetween NHA derived andT98G(cells from astrocytes adult in than higher is astrocytes actions PA on effects any have not did also tibolone hand, other the On affected. not was integrity mitochondrial trigg not did PA that finding the by explained be can differences These effect. any have not did tibolone with PA of combination the phosphorylation, in increase an induced alone tibolone although and that found We line. cell T98G the of those to conditions similar under treated were cells These brain). fetal from Tyr and expression we accordingly, interpreted be should results the and Article limitations some have might lines cell in mechanisms molecular of study the As compounds th with associated be could that mechanism a TERT, of expression the in involved gene or protein in was increase an without raloxifene, and estradiol receptors by TERT of phosphorylation estrogen of activation that demonstrated study another proteins HSP90 and Akt with association its by regulated is TERT of and proteins, other with TERT re therefore, of interaction promoting pathways other of induce might activation tibolone that suggest findings These death. cell increase not did inhibition ( duce cell death. Previously, a study demonstrated that the anti the that demonstrated study a Previously, death. cell duce 61 ) . er a strong effect on cell death cell on effect strong a er

, -

in contrast to contrast in 0 popoyain o oml ua atoye NA cls (derived cells (NHA) astrocyte human normal to phosphorylation 707 Fgr S (Figure 5

T98G, PA reduced Tyr reduced PA T98G, ) I sol b ntd ht amtc cd oxidation acid palmitic that noted be should It . ( 62 aimed

) in the NHA cells NHA the in . In this context, the assimilation of palmitic of assimilation the context, this In . eserved.

to extend our analysis of TERT protein protein TERT of analysis our extend to

- 707 phosphorylation in NHA cells, cells, NHA in phosphorylation 707 , therefore the the e protection by these these by protection e adult brain).adult - ( apoptotic function function apoptotic 60

it is possible that possible is it ) . Interestingly, Interestingly,

n fetal in ir

This article isThis article by protected copyright. All rightsr that observed we Additionally, depend will IL6 eff under by to exposed T98G for current the In neuroprotective in example, For induc the of depending and actions inflammatory in changes in located findings, these support expression i inhibition TERT in inhibition gene IL6 that demonstrated we experiments model our in expression the manner similar W L i a yoie ih litoi effects, pleiotropic with cytokine a is IL6 e previously observed that TERT and IL6 genes were regulated by regulated were genes IL6 and TERT that observed previously e

Acceptedcell other in especially ects, Article

a NF propidium iodide uptake test at test uptake iodide propidium chronic exposition to exposition chronic - kB factor factor kB its the y idn t a to binding by

of TERT inhibition TERT of cells under an inflammatory stimulus stimulus inflammatory an under cells

configuration cell thumb domain, which regulates regulates which domain, thumb ( ( 24 64

ncreased n ct isl o tamtc ri injury brain traumatic of insult acute an ) model ( on ) . 7 ,

PA ) as well as in as well as TERT is TERT ( . 63 hte atvto of activation whether considering that considering hs w have we Thus, , although higher although , , )

. we have observed that lower concentration lower that observed have we

Ti hptei ws orbrtd with corroborated was hypothesis This .

ing

In astrocyte cells, IL6 is increased to increased is IL6 cells, astrocyte In the reduction of reduction the PA ( motif 39

factor, this will be beneficial or detrimental or beneficial be will this factor,

required )

might

. IL6 induced a induced IL6 types like neurons like types in cells exposed to PA and tibolone and PA to exposed cells in

levels rsn in present cells

24 hours of hours 24 have

for BIBR1532 is an inhibitor that interact that inhibitor an is BIBR1532

exposed to exposed hypothesized and protein secretion were decreased by TERT TERT by decreased were secretion protein and concentrations did not increase not did concentrations

a the induction of induction the IL6 higher IL6 expression IL6 higher the

hc i ivle in involved is which trans eserved.

the the increase in increase secretion L gn DNA gene IL6 treatment ( LPS ( LPS 66 binding - ( inln ptwy i induced is pathways signaling 6 ) ht TERT that , .

7 Nonetheless, detrimental effects detrimental Nonetheless, an

) . by tibolone. by

. IL6 TERT neetnl, e bevd that observed we Interestingly, inductor of inflammation) inflammation) of inductor f TERT of These

it ( 6 a osre that observed was

carry out carry

) sequence

gene expression gene

, IL1 , ol be could with possib with . results Similar to other studies, other to Similar s

PA to both of IL6 are beneficial beneficial are IL6 of TERT re TERT β

the cell death cell

and TNF genes by genes TNF and

N o RNA or DNA

for the brain cells brain the for suggest that suggest and tibolone in a in tibolone and different actions, different

( pro 6 results euaig IL6 regulating s ) le

. with

- gulates IL6 gulates O detrimental

, n anti and r results ur

in T98G in assessed a motif a of the the of L is IL6 ( ( cells 67 65 by of ) ) - . . .

This article isThis article by protected copyright. All rightsr cellastrocyte functions andtheirpossiblefeedbackunder inflammatory stimuli. detrimental and beneficial under conditions. processes cellular distinct in participates telomerase that demonstrate results our Moreover, model. cell astrocytic an in activity and expression for show we conclusion, In possible therapeutic targets. might astrocytes of activation the of mechanisms the of elucidation diseases response astrocytic chronic a but brain, the in stress acute ameliorate oligodendrocytes and microglia neurons, as such types cell distinct with associated processes several regulate they because brain the in role crucial a play Astrocytes diseases. neurological of treatment con the in development drug for astrocytes targeting of potential the highlight results These compounds. estrogenic to exposure the to and PA with injury the an to in response involved processes cellular distinct in participates TERT that evidenced have We ne thatsuggests afeedback expression) and (activity cells.

Accepted Articleeded ET a rglt I6 expression IL6 regulate can TERT

to elucidate this hypothesis. ( 70 uue tde ae ne are studies Future ) . During astrocyte activation, cytokines as IL6 are increased. In this regard, the the regard, this In increased. are IL6 as cytokines activation, astrocyte During . ( 69 ) . On the other hand, activation of astrocytes is a defensive response to to response defensive a is astrocytes of activation hand, other the On . loop

hog NF through the between TERTIL6 and

first time how time first e ded

- to kB

define / and TT activation STAT3

PA , eserved. n un I turn, in

h seii role specific the and tibolone modulate telomerase protein telomerase modulate tibolone and

could existcould

L 6

a mdlt TERT modulate can

( .

68 H owever, future studiesare owever, future ) s .

of IL6 and TERT on on TERT and IL6 of provide insights about about insights provide Therefore, this result result this Therefore, s soitd with associated is

text of the the of text function This article isThis article by protected copyright. All rightsr PloSone. 2011;6(3):e17837. findings. correlation in majordepression: 13. medical sciences. and sciences Len ofTelomere analysis 12. heterochromatin. proteins and 11. 2016;629:241 stress reticulum to endoplasmic vulnerability 10. 2012;40(2):712 acts asahTR 9. a Molecular tyrosine 707. of family Srckinase via reversetranscriptase oftelomerase export nuclear 8. dise withNF loop in afeedback 7. NF 6. Gene. 2001;269(1 (hTERT). oftelomerase subunit 5. 2011;31(2):245 biology. and vascular thrombosis, Arteriosclerosis, inmacrophages. stimuli inflammatory by expression reversetranscriptase oftelomerase andinduction atherosclerosis activation in 4. 2008;26(5):454 and cells. Molecules cells. stem mesenchymal inhuman activity telomerase 3. genetics in Trends diseases. and genes, proteins 2. telomere 1. Reference The authors noconflictdeclare ofinterest. ofinterestConflict from (Grant No. Colciencias 824 Aplicados y YG Acknowledgments

Accepted Article kappaB - ase. Scientific reports. 2016;6:18685. reports. ase. Scientific G is supported by a PhD fellowship from fellowship PhD a by supported is G Wolkowitz OM, Mellon SH, Epel ES, Lin J, Dhabhar FS, Su Y, etal. SuY, FS, J, Dhabhar Lin ES, Epel SH, Mellon OM, Wolkowitz DA,Gonzalez Forero DNAdamage meet:telomerase, ends to make not Newways EH. SW,Blackburn Chan T, Naka Hosoi telomerase Human etal. DM, Gordon MG, Bonini MJ, P,Caron A,Green Reyes Sharma NK, triggers peroxide S.Hydrogen RP,Dimmeler Zeiher AM, J, Brandes J,Hoffmann Haendeler acts reversetranscriptase et Telomerase Huang C,al. XF, Li YH, Cheng Y, LiWX, Yang Wu XQ, directly regulates etTelomerase TD, al. Yan Shin EM, E, Khattar SC, G,Leow Saginc Ghosh A, JC, Poole HM,Zhao EB, Findeisen F,Heywood Gizard receptor Estrogen W,ParkKS. SJ, Seol Y, Cha Kwon M, Chalifa Rebhan MA. Martinez P,Blasco - binding proteins. Nature reviews Cancer.2011;11(3):161 reviews Nature proteins. binding -

dependen

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Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic ofthe catalytic gene regulation and Activity,function, TO. LG,Tollefsbol Andrews BA

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-- preliminary preliminary - 8.

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Accepted Article Q X,Ming Tao Sprague of astrocytes in induction Kim H. Telomerase Kim Y, H, Baek S, Bu Gonzalez C,Deighton Zhao Guzman CB, Hidalgo estrogenic aselectivetissue oftibolone: effects FD.Cardiovascular R,Marengo Campisi Gonzalez melaton effects of etal.Cytoprotective M, EA, Ling S, Gao J, Liu Wang D, Wang Z,Liu Gupta among lean, profile Freefattyacids Y, Li et al. Okekunle AP, LiC,DuS, C, R,Feng Luo H, et al. H,Kroger M, Soininen T, Kivipelto Rikkonen M, B,Tuppurainen Imtiaz oflong Effect JY. HR,Shim KimJH,Lee ImJA, Lee DC, KY Lee Shin YA, Wojcic O RM,etal. JA, Cawthon Taylor ChenH, LA, CG, DeRoo Parks Kim S, Jurk ofthe Annals stress. byoxidative shortening oftelomere Mechanism Oikawa S. S, Kawanishi u Y, Duan Z, Zhao F, Wei D, Zhang J, Tang B, et al. Telomerase reverse transcriptase reversetranscriptase Telomerase et al. B, Tang ZhangJ, D, F,Wei Zhao DuanZ, u Y, Neuroprotective effectso Neuroprotective with Stimulated Microglia in Inflammation and Damage Oxidative Reduces Tibolone gulator. gulator. d through Mechanisms Involving Estrogen Receptor Beta. Molecular neurobiology. 2017. neurobiology. Molecular Beta. Receptor Estrogen Involving Mechanisms through d D, Wilson C, Passos JF,OakleyF, Correia C,Passos Wilson D, - 78. -

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Kun Y,Wei . Low estrogen levels and obesity are associated with shorter telomere telomere with shorter associated are obesity and estrogenlevels . Low - 20. - alcoholic fatty liver disease patients: a case patients:a liverdisease fatty alcoholic

- Bin S,Hai Bin - Rodriguez M, Baez M, Rodriguez f the catalytic subunit of telomerase: A potential therapeutic therapeutic Apotential telomerase: of subunit catalytic f the

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- 45.

- Rodriguez M, Garcia M, Rodriguez -

control study. Lipids in Lipids study. control - - 45. 9. besity and weight gain in in gain andweight besity

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chain fatty acids fatty chain 71.

- in the mouse N20.1 mouse in the Year Period. Year Period. -

Dawley rat after rat after Dawley Tibolone Tibolone - Segura in on - ere Segura Segura - - 46.

This article isThis article by protected copyright. All rightsr 2008;3(6):1101 Nature protocols. 51. 2007;2(9):e898. PloSone. genes. ofhousekeeping based selection 50. 2009;37(6):e45. acids research. Nucleic PCRdata. quantitative of the analysis bias in and baseline linking efficiency: Amplification 49. 2006;1(3):1559 protocols. 48. 2015;94(2):305 J Genet. sample. with a f associated 47. RFLP studies. for blood from 46. research. 2009;37(3):e21. Nucleic acids PCR method. 45. 2018;45(4):633 reports. biology Molecular qPCR. 44. 2001;47(3):519 Chem. activity. Clin telomerase the detection of for assay protocol 43. pro Nature protocol. amplification repeat the telomeric using activity 42. 2003;55(2):61 Cytology. Analytical Cytomet events. singlecellsignaling monitoring 41. 2018;70(4):1143 Cytotechnology. LN18 cells. tra reverse telomerase human of 40. 2015;23(10):1934 Structure. BIBR1532. Specific theHighly by Inhibition Telomerase 39. 2018. immunology. of Journal Gene Expression. Muscle Chemokine 38. 2017. neurobiology. Molecular Cell Astrocytic in Morphology andCell Functionality Mitochondrial 37. features. ofcell characterization 36. 2017. neurobiology. Molecular Assay. a Scratch Under (T98G) Model Astrocyte Human Prot Reduces Neuroglobin 35. 2016;433:35 expression. neuroglobin of upregulation andthe beta receptor amechani through deprivation glucose from cells Tibolone protects astrocytic 34. 1996;156(6):2247 ofco characterization 33. 1979;99(1):43 physiology. cellular of Journal vitro. arrest G1 in phase 32.

Accepted Article Schmittgen TD, Livak KJ. Analyzing real Livak KJ. Analyzing TD, Schmittgen F, Gerbens RM, Hofstra ES, de RS, Bont Fehrmann HJ, de Jonge C, Ramakers JM, Ruijter real using mRNA of SA. Quantification Bustin RE, T,Nolan Hands Speck Lahiri DK,Nu multiplex quantitative monochrome anovel by measurement length Telomere RM. Cawthon master of DA.Effect Forero Jimenez KM, Real Gruber A. M, D,Bjorkholm Xu M, Hou telomerase of detection Nonradioactive JW. WE,Shay Wright AE, BS,Hochreiter Herbert phospho GP.Intracellular Nolan Krutzik PO, regulation GK.Down Chetan MM, Bharath MK, Srinivas MM,Sibin C,Venkataswamy Lavanya M Harkisheimer H, Hoffman C, Rice C, Bryan TNF for Required Are and TERT PINX1 AJ. K, Deacon Knox Gonzalez Molecular R, etal. E,Dedecker Bruyneel F, I,Lefranc Rorive S,Doyen Belot N, Baez Avila and MP, etIdentification al. Dierich S, Singhrao MT, C,Schouft Mauger P, Gasque P,Chan ancho T98G: Stein GH. an Glia. 2001;36(3):375 Glia. - - - 46. Jurado E, Vega GG, Aliev G, Tarasov VV, Esquinas P, Echeverria V, et al. V,et Echeverria P, Esquinas VV, G,Tarasov Aliev E, VegaGG, Jurado Rodriguez M, Garcia M, Rodriguez - Hernandez CA, Ojeda DA, Castro DA, Ojeda CA, Hernandez

- Giraldo Y,Garcia Giraldo - 55. unctional polymorphism in the monoamine oxidase A gene in a South American American aSouth A gene in oxidase monoamine inthe polymorphism unctional rnberger JI, Jr. A rapid non A JI,Jr. rapid rnberger

mplement C3 receptors on human astrocytes. astrocytes. onhuman receptors C3 mplement

substratum attachments in human glial tumors relates to prognostic prognostic relatesto tumors glial inhuman attachments substratum - ection of Conditioned Medium from Human Mesenchymal Stem Cells in Cells Stem Mesenchymal Human Medium from ofConditioned ection 82. Nucleic acids research. 1991;19(19):5444. research. Nucleic acids

- 90.

Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ,et al. denHoff O, van WM,KarlenY,Bakker Hoogaars - nscriptase (hTERT) expression by BIBR1532 in human glioblastoma glioblastoma human in BIBR1532 by expression (hTERT) nscriptase 8. rage -

- 8. - - Segura LM, Hidalgo Segura LM,

70. Segura LM, Echeverria V, Barreto GE. Barreto V, Echeverria Segura LM,

independent human tumor cell line that exhibits stationary stationary thatexhibits cell line tumor human independent - 54. - - - time PCR data by the comparative C(T) method. method. C(T) comparative bythe PCRdata time Vega LJ, Forero DA. Forero Vega LJ, ry Part A : the journal of the International Society for Society theInternational of A thejournal : ry Part enzymatic method for the preparation of HMW DNA DNA HMW of the preparation for method enzymatic

- mixes on the measurement of telomere lengthby telomere of measurement onthe mixes 8. - time quantitative telomeric repeat amplification amplification repeat telomeric quantitative time -

, Sweeney M, Skordalakes E. Structural Basis of Basis E.Structural M,Skordalakes Sweeney , protein staining techniques for flow cytometry: cytometry: flow for techniques staining protein eserved. - Lanussa O, Baez E, Gonzalez J, Barreto GE. GE. J,Barreto Gonzalez O,E, Baez Lanussa

Molecular and cellular endocrinology. endocrinology. cellular and Molecular - alpha

s Treated with Palmitic Acid. Palmitic Acid. with s Treated

Relative telomere length is length telomere Relative Journal of immunology. ofimmunology. Journal - tocols. 2006;1(3):1583 tocols. 54.

Kamps WA, et al. Evidence Evidence WA,et al. Kamps - Induced Airway Smooth Smooth Airway Induced - Tibolone Preserves Preserves Tibolone

time RT time sm involving estrogen estrogen involving sm - PCR. Nature PCR. - 24. - 42. Blockade of Blockade

- 90.

This article isThis article by protected copyright. All rightsr 2014;94(4):1077 reviews. Physiological 70. 2010;7(4):338 NeuroTherapeutics. Experimental for Society oftheAmerican : journal the targets. Neurotherapeutics 69. 2017;2017:5958429. of Mediators Cells. Cancer inColorectal Signaling the AInhibited Withaferin TNF 6 and 68. PtA):1218 acta. 2016;1863(6 etbiophysica Biochimica 67. 2012;1822(6):831 acta. biophysica circuitry imbalance neuronal causes elevation 66. secreted IL 65. 2003;181(2):130 IL of expression 64. Sup 2006;8 63. 1976;52(1):161 biology. Developmental brain. developing by oxidation 62. 2004;183(3):605 endocrinology. Journal of The cascade. activityviaAkt oftelomerase cells activation by PC12 transfected beta against raloxifene protect 61. 2003;536(1 andanti activity 60. 7):1046 cell2008;121(Pt of science. Journal stress. underoxidative function mitochondrial but protects telomere shortening counteract 59. 2012;515(1):39 hypoxia after rats neonatal in mechanism antiapoptotic 58. with oxygen neurons TERT in 57. 2002;66(3):407 MMBR. : reviews biology molecular 56. by linear 55. & 54. 97. Akt and localization signal 53. 2009;29(6):929 biology. andvascular thrombosis, Arteriosclerosis, damage. from function DNAand mitochondrial protects to and binds transcriptase reverse telomerase 52. cell. 2011;2(9):726 cell.

Accepted Article Pekny M, Pekny therapeutic as potential and their astrocytes of M.Functions Nedergaard Kimelberg HK, Chung M, Becker Rothaug IL WT, etal.Brain M,Brown Malik SheikhAM, DP, McCloskey KK, Wei H, Chadman M, Y, Q, Hu X,Wang Li Jia Li Sun L, Y, Astrocyte et J,al. J, Hernandez Carrasco N,J, Camats M, M,Giralt Lago Penkowa T.Interleukin Kishimoto Ter JB, Warshaw et Igarashi H, a C, J,Ohshima K,Kawagoe M,Takahashi Ohmichi Du B, J J,Hoffmann Haendeler A an via ofTERT role neuroprotective D.The Q,Mu Z,Ye T, Duan Y,Xiong F,Qu Zhao of mechanisms roleand neuroprotective The L, et F,Luo al. Zhao L, Zhang D, Li J,QuY,Chen JW. Hum WE, Shay YS, Wright Cong telomerase of Inhibition F,et al. H,Sugawara H, Yoshida Takahashi T, N, Kasai M, Ueno Oda Protein aging. activity in telomerase of regulation Molecular JP. JQ, H, Wang Liu Nicholls C,Li IK.Nucl P,Chung J, Khadka Chung N,Ja S,Buchner J,Drose Haendeler - hmed S, Passos JF, Birket MJ, Beckmann T, Brings S, Peters H, et al. Telomerase does not does not Telomerase Peters H,et al. S, T, Brings MJ, JF,BirketBeckmann hmed S,Passos chain fatty acids: a structural analysis. The Biochemical journal. 2002;367(Pt 2):329 2002;367(Pt journal. Biochemical The analysis. a structural acids: fatty chain - alpha Increased Telomerase Activity through NF Activity through Telomerase Increased alpha - pl 2:S2. pl - 6 in acute neuroinflammation. Oncotarget. 2017;8(25):40065 Oncotarget. neuroinflammation. in acute 6 3):180

SS, Wu Y, Okobi Q, Adekoya D, Atefi M, Clarke O, et al. O, et al. M,Clarke Atefi D, Q,Adekoya Okobi Y, SS, Wu - - - 43. 6 protects the CNS against a focal brain injury. injury. brain afocal against the CNS protects 6 - apoptotic function by protein by function apoptotic 48. Pekna M. Astrocyte reactivity and reactive astrogliosis: costs and benefits. andbenefits. costs astrogliosis: and reactive reactivity M.Astrocyte Pekna

38. ry ML. Cellular energy metabolism during fetal development. VI. Fatty acid Fatty VI. development. during fetal metabolism energy ML. ry Cellular

- mediated phosphorylation. Journal of cell science. 2012;125(Pt 11):2684 2012;125(Pt cell science. of Journal phosphorylation. mediated - Pauly C,Rose Pauly - glucose - amyloid - - , Rahman S, Zeiher AM, Dimmeler S. Regulation of telomerase telomerase of S. Regulation AM,Dimmeler S,Zeiher Rahman , 6: discovery of a pleiotropic cytokine. Arthritis research & therapy. &therapy. research cytokine. Arthritis a pleiotropic of discovery 6: 15. - - 42. 53. -

53.

deprivation. Neuroscience. 2013;252:346 Neuroscience. deprivation.

- 98. induced neurotoxicity in estrogen receptor alpha receptor inestrogen neurotoxicity induced - ear import of hTERT requires a bipartite nuclear bipartite nuclear a hTERT requires of ear import John S. The roleofinterleukin S.The John

kob S, Altschmied J, Goy C, et al. Mitochondrial Mitochondrial al. J,GoyC,et S, Altschmied kob an telomerase and its regulation. Microbiology and and Microbiology its and regulation. telomerase an

et al. al. et s and mediatesautism s and - protein interaction and phosphorylation. FEBS letters. FEBS and phosphorylation. interaction protein Neuroprotection byIFN Neuroprotection - 25, table of contents. table of 25, eserved. - 27. - ischemia brain injury. Neuroscience letters. letters. Neuroscience brain injury. ischemia

- kappaB/STAT1/STAT3 Activation, and Activation, kappaB/STAT1/STAT3

Experimental neurology. neurology. Experimental - like behaviors. Biochimica et Biochimica like behaviors. Proinflammatory Cytokines IL Cytokines Proinflammatory - 6. - 6 signaling in nervous tissue. nervous signaling in 6 -

78. - gamma via astrocyte gamma via - 35. inflammation. inflammation.

l. Both estrogen and and Both estrogen l. -

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- targeted targeted - - 6 - 34. -

- - This article isThis article by protected copyright. All rightsr (C) Accepted(A) for: (a.u.) units arbitrary as represented fluorescence Mean PA. mM 1 adding before tibolone production. 3. Figure TIB: tibolone, acid. PA:Palmitic compari multiple Tukey's one by A followed experiments. ANOVA independent three least at of SEM ± and Mean represents graph qPCR. by measured Telom next and performed, was isolation DNA and protein then, stimuli, acid Palmitic mM 1 of hours 24 by followed tibolone µM 70 with hours cells. T98G in length telomere and activity Telomerase 2. Figure Palmitic acid. tibol TIB: <0.05. *: and <0.01 **: <0.001, represents: *** graph the In analysis. Two experiments. independent three least at of SEM ± Mean represents graph Bar carnitine. mM experiments, all For hours. 48 and 36 24, 12, for acid Palmitic mM 1 with treatment a by followed tibolone µM 70 with hours 24 for Articletibolone µM normalized software cytometry flow the using tibolone. with pretreated cells (Blue). tibolone with or (Red) alone hours, 36 at PA with treated cells in (Green) control the to respect expression protein ri the to for to out carried was analysis cytometry determine flow Then, hours. 48 and 36 24, 12, for treatment treatments. 1. Figure Figure legends

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**** . - - 227 227 707 re re This article isThis article by protected copyright. All rightsr used was test forstatistical analysis. TIB:tibolone, PA:Palmitic acid. three least at one of A SEM experiments. ± independent and Mean represents graph Bar carnitine. 2mM and BSA % 2.5 data cytometry flow for plot representative data. cytometry hours (24 analysis. cytometry flow for antibodies the with incubated and fixed were cells Next, hours. 24 for PA mM 1 by followed hours 24 for acid palmitic to exposed Tyr Telomerase 8. Figure wasusedcomparisons test forstatistical analysis. TIB:tibolone, PA:Palmitic acid. inde at three of SEM least ± and Mean represents graph Bar carnitine. 2mM and BSA % 2.5 in consisted its PA. and tibolone hours). (48 acid palmitic and hours) analysis. cytometry flow for Nex hours. 48 for PA mM to 1 by followed exposed astrocytes human normal in tibolone and acid palmitic expression protein Telomerase 7. Figure independent three for statis least at of SEM ± one and A experiments. Mean represents graph Bar carnitine. 2mM flo of Ser for analysis

Accepted Article (D) w cytometry data. For all experiments, control sample consisted in 2.5 % BSA and and BSA % 2.5 in consisted sample control experiments, all For data. cytometry w ersnaie lt f lw yoer data. cytometry flow of plot representative tical TIB: analysis. tibolone, PA:Palmitic acid. ) and palmitic acid (24 hours) treatments, and its its and treatments, hours) (24 acid palmitic and ) -

edn eprmns A one A experiments. pendent 227 in cells treated with DMSO and tibolone, and its and tibolone, and DMSO with treated cells in 227 (C)

(C) - way ANOVA followed by Tukey's multiple comparisons test was used was test comparisons multiple Tukey's by followed ANOVA way Tyr T ERT protein expression in cells treated with DMSO and tibolone, and and tibolone, and DMSO with treated cells in expression protein ERT -

0 popoyain nlss o tbln v DS, n its and DMSO, vs tibolone for analysis phosphorylation 707 and tibolone and . Cells were treated with 20 µM and 10 nM Tibolone for 24 hours hours 24 for Tibolone nM 10 and µM 20 with treated were Cells . - (A)

707 phosphorylation status in normal human astrocytes astrocytes human normal in status phosphorylation 707 - TERT protein expression in cells treated with tibolone (24 tibolone with treated cells in expression protein TERT a AOA olwd y ue' mlil comparisons multiple Tukey's by followed ANOVA way (B) . Cells were treated with 20 µM and 10 nM Tibolone Tibolone nM 10 and µM 20 with treated were Cells . t, cells were fixed and incubated with the antibodies antibodies the with incubated and fixed were cells t,

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(B) ersnaie plot representative

(D)

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(D) (D) This article isThis article by protected copyright. All rightsr Accepted Article eserved.

This article isThis article by protected copyright. All rightsr Accepted Article eserved.