What Are Microarrays Teaching Us About Sleep

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31.05.2013 What are microarrays teaching us about sleep?

www.ncbi.nlm.nih.gov/pmc/articles/PMC2942088/ 1/14

What are microarrays teaching us about sleep?

Miroslaw Mackiewicz, John E. Zimmerman, [...], and Allan I. Pack

Abstract

Man y fundam ental questions about sleep rem ain u nan swered. The presence of sleep acr oss phy la

suggests tha t it mu st serv e a basic cellula r an d/or molecular fun ction. Microarr ay studies,

perform ed in sev eral m odel sy stems, ha v e identified classes of genes that ar e sleep-state regu lated.

This ha s led to the followin g concepts: first, a function of sleep is to m ainta in sy na ptic hom eostasis;

second, sleep is a stage of ma crom olecule biosy nth esis; th ird, exten ding wa kefulness leads to

down regulat ion of sev eral im portan t m etabolic pathwa y s; and, four th, extending wa kefulness leads

to endoplasmic reticu lum stress. In hu m an studies, m icroarra y s ar e being a pplied to the

identification of biomar kers for sleepiness and for the comm on debilitating condition of obstru ctiv e

sleep apnea .

High-throughput approaches to study gene expression

The search for genes inv olv ed in regu lation by , and of, sleep an d wak efuln ess and the quest to

un derstand the fun ctions of sleep at th e molecular lev el began a decade before th e adv ent of

m icroarra y s. Sev eral can didate genes w ere stu died to elucidate their roles in sleep an d wakefuln ess

(for a rev iew, see [1 ]) . Subtract iv e hy bridizat ion carr ied out on br ain m RNA from sleep-depriv ed

rats , perform ed in th e early 1 99 0s, was the f irst high-through put attem pt to identi fy such genes

[2 ]. Later, the a pplication of differen tial display [3 ] broadened the candidate gene and subtract iv e

hy bridization approaches to screen for genes wh ose fun ctions w ere poorly cha ra cterized or

previously u n known.

A s a r esu lt of sequ en cin g efforts, ther e has been an exponential gr ow th in th e am oun t of inform ation

av ailable about the DNA sequen ce of the hu m an g enome (e.g., see [4 ]) as well as th e genom es of

v a r iou s m ode l or g a n ism s ( e. g ., see [5 ,6 ]). Consequen tly , th ousands of genes hav e been discover ed,

including m any novel genes for w hich sequences were una v ai lable prev iously . Th e role of man y of

these genes is un known . This facilitated the adv ent of m icroarr ay approaches.

A m ic r oa r r a y con ta in s t en s of th ou sa n ds of g en es a n d is a tool for sim u lta n eou s m ea su r em en t of

their expr ession. Sign ifican t adv an ces in gen e ann otations, progress in dev elopm ent an d

standar dization of micr oar ra y platform s, as w ell as the expansion of data an aly sis tools, inclu ding

new an d power ful statistical approaches to assess expression data (rev iewed in [ 7 ,8 ]) , make a

m icroarr ay approach th e ideal way to ini t ia l ly determ ine the chang es in tran scription of the

genom e in r esponse to, or as a consequen ce of, sleep and/or wa ke state. Howev er, m icroarra y s

describe what m ight h appen r ather than wh at does happen in a cel l or t issue because changes in a

giv en m RNA m ight n ot tra nslate into changes in the r elev ant protein. Microarr ay s are a tool of

discover y an d, as such , results are hy pothesis-gener ating . Ther efore, hy potheses ar ising from

m icroarra y studies need to be fur ther assessed. In par ticula r, i t is im porta nt to disting uish w heth er

cha ng es in gen e expression a re being driv en by sleep v ersus driv ing sleep or w akefulness. Nov el

pathw ay s are, howev er, being identi fied and, i f conf irmed, w i l l provide new targets for t herapeutic

interv ention to address problems w ith sleep.

Microarrays in sleep research

Microarr ay s offer a new w indow on th e difference betw een the sleeping an d awa ke brain a nd hold

th e promise of facilit at ing a nsw ers to som e of the m ajor questions in sleep biology (Box 1 ; see also

[9 1 1 ]) .
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Box 1. Major questions in sleep biology

Wh a t a r e th e fu n ct ion s of slee p?

Wh ic h g en es r eg u la te slee p a n d w a kefu ln ess?

Wh a t m ole cu la r ch a n g es set th e du r a tion of w a kefu ln ess th a t ca n be su sta in ed w it h ou t

i m pa i rm ent?

Wh a t a r e th e m olec u la r con sequ en ces of sleep de pr iv a tion ?

Wh a t is th e ba sis of in di v idu a l di ffe r en ces in r espon se to sl ee p de pr iv a tion a n d oth er sleep

characteristics?

A r e th er e biom a r ker s for slee p dr iv e a n d for spec ific sleep di sor der s?

These questions ar e relev ant to the perv asive problem of sleep depriv ation in industrialized societies

an d to the high prev alence of sev eral sleep disorders [1 2 ] . In th is rev iew, w e describe what w e hav e

learn ed from m icroarr ay experim ents designed to address these questions and discuss futu re

opportu nities for the application of these an d r elated approach es.

Microarr ay studies addressing sleep fun ction h av e focused on t he bra in in v ar ious species [1 3 1 9 ],

a lthough a r ecent study exam ined other organs [1 8 ]. Assessm ent of the tr anscriptome in m odel

org an ism s ha s demonstrat ed chang es in the lev el of tra nscripts of man y genes betw een sleep an d

w a kefu ln ess; a s m a n y a s 1 0 % of g en es in m ou se br a in ch a n g e th ei r ex pr ession bet w ee n th ese tw o

be h a v ior a l sta tes [1 7 ]. Microarra y studies perform ed to date ha v e used different plat form s and

differen t study designs (Figur e 1 ). Som e stu dies hav e ev alu ated genes cha ngin g expression between

sleep and w akefulness using fold cha ng es (e.g., see [1 3 ]), wh ereas other s hav e used false-discov ery

ra te strateg ies (e.g. see [1 7 ]). Despite these differences, ther e is a similar ity in th e results with

respect to path w ay s in the bra in tha t are affected by sleep and wa kefulness (Table 1 ) . There

rem ains debate as to the num ber of genes that a re expressed differentia lly between sleep an d

w a kefu ln ess ( e. g ., see [1 3 ,1 4 ,1 7 ,1 9 ]). This probably reflects differen t study designs an d, in

particu lar, the sam ple sizes (power) u sed in different studies [1 3 ,1 4 ,1 7 ,1 9 ].

Figure 1

Examples of experi mental strategies in micr oarray r esearch t o eluc idate the identi ty of genes

w hose ex pr essi on ex hi bi ts di f fer ences b et w een b out s of sl eep and w ak ef ul ne ss. The se

complementary strategies address the confounding effects of sleep depri vati on-induced ...

Table 1

Micr oarr ay appr oaches to study gene expr ession during sleep, wakeful ness or sleep depriv ation

and the key functi onal c ategori es of genes expr essed dif ferenti all y among behavi oral states

Model systems for the study of sleep

In r ecent y ears, an im porta nt a dv an ce in sleep research h as been th e identification of a sleep state

in non-m am m alian m odel systems: nam ely, the frui t f ly (Dros oph ila m ela no ga s ter) [2 02 2 ], the

zebrafish (Danio rerio ) [2 3 2 5 ] and, m ost recently , the nem atode (Caenorhabditis elegans ) [2 6 ] (see

al so [2 7 2 9 ]). Cru cial to the identification of sleep in th ese m odel system s has been th e use of

be h a v ior a l r a th er th a n el ec tr oph y siolog ic a l cr it er ia to def in e th e slee p sta te. Th e m a in beh a v ior a lcriteria , in addition to quiescence (lack of mov em ent), ar e the follow ing: f irst, elev ated ar ousal

threshold ( i.e . when asleep, i t takes a larger st imu lus to m ake the an imal m ov e or a longer t ime to

respond t o a f ixed stimu lus); second, homeostasis (i.e. follow ing sleep depriv ation th e an im al

ret ur ns to sleep faster, h as incr eased dura tions of sleep bout s, often considered a m easur e of sleep

a
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depth, an d can sleep at inappropriate circadian tim es); and, th ird, the tim ing of sleep propensity

correlates with th e molecular clock or rhy thm ic expression of clock genes. Not only ar e the

be h a v ior a l a spec ts of slee p con ser v ed a m on g th e m ode ls bu t th er e is con ser v a tion of b oth

neur otran smitter systems and the com ponents of the molecular s ignal ing pathw ay s that regu late

sleep [3 0]. For exam ple, s ignal ing m echanism s inv olv ing cy cl ic AMP, w hich promotes wa kefulness

[3 1 ,3 2 ], and epiderm al gr ow th factor, w hich pr om otes sleep [3 3 ,3 4 ], hav e the sam e role in

differen t m odel sy stems (for r ev iew, see [3 0]) .

Model sy stems provide a gr eat opportu nity for inv est igation by m icroarr ay s. Microarr ay s hav e

be en a pp li ed to t h e stu dy of sle ep in fr u it fl ies [ 1 4 ,1 9 ], rat s [1 3 ,1 5 ] and m ice [1 7 ,1 8 ]. In a ddition,

m icroarr ay studies hav e been conducted recently in the w hite-crowned sparrow (Zo no trich ia

leukophrys) [1 6 ], albeit with a lim ited sam ple size. This spar row, like ma ny long -distan ce av ian

m igrants , has the abi l i ty to go without s leep during i ts migr ation. A n a rea ripe for futu re

inv estigation is microarr ay studies of cha ng es in gen e expression in the n em atode or zebrafish.

Microarr ay studies tha t ha v e been conduct ed in f lies, rodents and birds show th at similar m olecular

pathw ay s ar e altered by sleep, wa kefulness an d sleep depriv ation (Table 1 ).

What microarrays are teaching us about the molecular functions of sleep

A n a ly ses of th e fu n ction a l ca teg or ies of g en es ch a n g in g ex pr ession be tw een sleep a n d w a kefu ln ess ,

as rev ealed by m icroarra y studies, hav e led to new hy potheses about the fun ctions of sleep an d are

now th e focus of hy pothesis-driv en research. Tw o m ajor theories ha v e emerg ed that ar e not

m utu al ly exclusiv e: fi rst , th at s leep and w akefulness regulate sy naptic strength, w ith u p-scal ing

dur ing w akefulness an d dow n-scaling du ring sleep [3 5 ,3 6 ]; an d, second, t ha t sleep is a stage of

m acr om olecule biosyn thesis [1 7 ]. In t he follow ing sections, w e explore each of th ese th eories.

Sleep and wakefulness and synaptic scaling

Microarr ay studies condu cted in rats [1 3 ] identified nu m erous genes inv olv ed in th e acquisition an d

potentiat ion of syn aptic plasticity (a term relatin g to the ability of syn apses to cha nge in streng th);

these genes expressed increased tran script lev els dur ing w akefulness. Conv ersely, t he lev els of

expression of genes inv olv ed in sy naptic consolidation or depression incr ease durin g sleep [1 3 ].

These observ ations hav e been incorpora ted with in a hy pothesis of sleep fun ction r eferr ed to as the

sy na ptic h om eostasis theory of sleepw ake c ontr ol [ 3 5 ] (for a rev iew of this theory , see [3 6 ]). It is

postulated tha t w akefulness is accompa nied by sy na ptic potentiat ion of cortical netw orks thr ough

br a in -der iv ed n eu r otr oph ic fa ctor (BDNF)-depe n den t a n d oth er sig n a li n g m ec h a n ism s. Su ch

sy naptic potentiat ion is achiev ed through act iv i t ies and learning during wa kefulness (com m only

term ed as experience) and occurs in neur onal circui ts that ar e act iv ated by t he releva nt

experience. Indeed, stimu lation of wh iskers in th e ra t m odifies the local electroencephalogra m

(EEG) pattern (the EEG is a m easure of electrical activ ity produced by the bra in) [3 7 ,3 8 ]. This

observ ation supports the n otion th at th ere is a potentiation of syn aptic strengt h in the bar rel cortex

tha t receiv es and processes tact i le inform ation deriv ed from t he contra latera l face of the an im al. It

is postulated tha t an incr ease in over all sy na ptic potentia tion dur ing w akefulness will require more

resour ces (energ y , space) to main tain t his lev el of potentiat ion. It has been furt her sug gested tha t

syn aptic potentiat ion is l inked causally to the inten sity of EEG slow-wa v e activ ity dur ing

subsequen t sleep and tha t, dur ing slow-wa v e sleep, ther e is syn aptic down -scaling. Such dow n-

scal ing of sy naptic strength has a beneficia l ef fect on n euronal function by retur ning the sy stem to

an ov erall pre-wakefulness balance, alth ough t his leav es tra ces of experiences that occurr ed

dur ing w akefulness and incr eases syn apse signa l-to-noise r atios.

Sleep and wakefulness and macromolecule biosynthesis

Microarr ay studies hav e also identified other classes of genes w hose patter ns of expression cha ng e
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dur ing sleep [1 7 ]. In m ouse cerebral cortex a nd, to a lesser extent , hy potha lam us there is

upregulat ion dur ing s leep of genes encoding proteins in v arious, predom inantly interm ediary ,

bi osy n th et ic pa th w a y s for h em e, pr otei n a n d li pi d [1 7 ]. A significan t nu m ber of genes encoding th e

struct ur al constitu ents of the ribosom es, tra nslation-regu lation activ ity an d form ation of tran sfer

RNA (tRNA; a sma ll RNA th at t ran sfers a specific am ino acid to a gr owing poly peptide durin g

protein syn thesis) an d ribosom e biogenesis ar e also upr egula ted dur ing sleep. Thu s, sy nth esis of

proteins and other m acr omolecules seems likely to occur dur ing sleep, m ore so tha n du ring

w a kefu ln ess.

Genes wh ose expression in creases progressiv ely dur ing sleep inclu de genes encoding m ultiple

enzym es of the cholesterol-syn thesis pathw ay , proteins inv olv ed in ch olesterol uptake an d tran sport

an d relev an t tr an scription factors and cha perones responsible for tr an scriptiona l regu lation of

ch olester ol-rela ted gen es [1 7 ]. Thu s, mem bran e cholesterol is expected to increa se dur ing sleep.

Cholesterol has an important r ole in m embra ne stabil i ty and is the key stru ctur al com ponent of

m em bran e micr odom ains called lipid rafts [3 9 ]. The tr an script levels of sev eral g enes encoding

lipid-ra ft-resident pr oteins, suc h a s flotillin , also incr ease dur ing sleep [1 7 ]. Rafts bring together

neu rotran smitter r eceptors with other sign aling m olecules an d, by so doing, alter th e stren gth of

signal ing [3 9 ]. The r eassem bling of l ipid ra fts dur ing sleep w ould alt er signaling of sev eral

neur otran smitters [4 0 4 3 ]. This migh t be in prepar ation for subsequen t wa kefulness, wh en ther e

is enh an ced release of v ar ious neu rotran smitter s on a rousal from sleep. Reassem bly of l ipid ra fts

during s leep would enable max imal s ignal ing on aw akening. I t is unclear h ow an increase in

cholesterol sy nth esis dur ing sleep would im pact on syn aptic down -scaling dur ing sleep tha t is

proposed in th e sy na ptic hom eosta sis th eory of sleepw ake contr ol [3 5 ].

In a ddition, du ring sleep there is an upr egula tion of genes encoding pr oteins inv olv ed in the

funct ionin g of v esicle pools, as well as in th e functioning of all antioxidant enzy m es an d com ponent s

of intrac ellular tra nsport. T hese observ ations sug gest that sleep is a stage of rebu ilding an d repair

in pr eparat ion for subsequen t w akefulness (Figur e 2 ).

Figure 2

During wakef ulness, upregul ation of genes inv olved in synaptic p lastici ty occur s and, wi th

extended wakef ulness, upregul ation of molecul ar c haperones foll ows. If w akeful ness is ex tended, a

major mechanism promoting sleep might be the progressiv e decli ne ...

What microarrays are teaching us about mechanisms limiting the duration of wakefulness

In h um ans, wa kefulness can be sustained for 1 6 h w ithout impairm ent of perform ance [ 4 4 ]. In

m ice, the dur ation of the longest episodes of sustained wa kefulness is mu ch shorter, being in the

order of 3 h [4 5 ,4 6 ]. Accordingly , there m ust be a cost to extending wa kefulness an d there is l ikely

to be molecular m echan ism s tha t lim it the dur ation of wakefulness. If so, extending w akefulness

be y on d th ese li m it s, a s i n a cu te sleep de pr iv a tion , sh ou ld be de le ter iou s. Mic r oa r r a y st u di es h a v e

rev ealed two key concepts: first that extending w akefulness leads to endoplasmic r eticulu m (ER)

stress; and, second, tha t m ult iple genes in key cel lular pathw ay s are down regulated w ith prolonged

w a kefu ln ess.

Extended wakefulness leads to cellular stress

Studies of gene expr ession ha v e rev ealed tha t expr ession of the HSPA5(heat sho ck 70 kDA protein 5)

gene, w hich encodes the m olecular cha perone com m only kn ow n as BiP, increases w ith extended

w a kefu ln ess i n di ffe r en t spec ies a n d di ffe r en t br a in r eg ion s [ 1 3 ,1 6 ,1 7 ,4 7 ] (see a lso [4 8 ,4 9 ]). Th ese

finding s sug gest tha t, w ith ext ended wa kefulness, th ere is stress in t he ER. In r odents, other genes
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encoding heat -shock proteins an d m olecular cha perones are also upr egula ted durin g sleep

depriv ation [1 7 ,4 9 ]. Increa sed expr ession of th e Hs pa5gene occur s as part of the signa ling path w ay

called th e u nfolded-protein response (UPR). This is an adaptiv e response tha t en ables cells to sur v iv e

stress in th e ER, resultin g from pert ur bations in ca lcium homeostasis, redox statu s, elev ated

secretory protein sy nth esis, m isfolded proteins, glu cose depriv ation or altered gly cosy lation [5 0].

The UPR helps to restore norm al ER funct ion by reducin g protein tran slation an d by u pregu lating

th e expression of ch aper ones to increa se th e ER capa city for folding or to prom ote degr ada tion of

m isfolded proteins (for r ev iews, see [5 1 ,5 2 ]). All com ponent s tha t ar e activ ated as part of the UPR

ar e elev ated in m ouse cerebral cortex after 6 h of sleep depriv ation [4 8 ]. In t his study , sleep

depriv ation w as perform ed at the begin ning of the ligh ts-on period, thus prolong ing w akefulness

tha t norm ally occu rs in the lights-off period. A recent m icroarr ay study indicates that expression of

the Hs pa5gene incr eases with sleep depriv ation not only in bra in but a lso in liv er [1 8 ]. Th us, ER

stress migh t be a g enera l response to sleep depriv ation th at is not specific to brain. Hen ce, the

prev ailing eth os that sleep is of the brain, by the bra in an d for th e brain [5 3 ] is challenged by

recent m i croa rra y da ta [1 8 ]. There is, howev er, l imited inform ation cu rrently on h ow s leep and

prolong ed wa kefulness im pact on gen e expression in peripheral t issues an ar ea ripe for fut ur e

study .

Downregulation of genes for multiple cellular processes as wakefulness is prolonged

In both fruit-fly brain s an d mouse cerebra l cortex a nd hy potha lam us, the larg est class of genes that

ar e expressed differentially between sleep-depriv ed anim als and sleeping c ontr ols are th ose tha t

show decline in expression w ith pr olong ed wa kefulness [1 7 ,1 9 ]. As the dur ation of wakefuln ess

progresses, th ere is a reduct ion in the expr ession of genes inv olv ed in m ultiple phy siological

processes; reduct ion in th ese processes mig ht act together t o lim it w akefulness. In the brain of the

fruit f ly , m ultiple genes inv olv ed in different steps in the protein-product ion path w ay ar e

down regulated w ith extended w akefulness [1 9 ] . In m ouse cerebral cortex and hy pothalam us, there

is a reduction dur ing ex tended wa kefulness in th e expression of genes encoding proteins inv olv ed in

the m ain pathw ay s of carbohy drate, energy , tr icarboxy l ic acid (TCA) anabolism and v arious

m etabolic pathway s, such as l ipid, a ldehy de and am ine sy nthesis [1 7 ] . Thus, a m echanism

promoting sleep mig ht be th e decline in processes tha t help sustain wa kefulness, thereby enabling

sleep to occur (Figur e 2 ).

Genetic perturbations can test the functional roles of genes identified using microarrays

One disadv an tag e of microarr ay strategies is that one does not know w heth er th e genes identified as

cha ng ing expr ession with behav iora l states are doing so as a consequen ce of the state (they could be

part of the fun ction of sleep) or w heth er th ey could be inv olv ed in r egula ting t he sleep state. Th ese

possibilities ar e not m ut ua lly exclusiv e.

This disadv an tag e has led researcher s to use the forw ar d-genetic strateg y of screening m ut ant s for

an a ltered sleep phenoty pe. If a m utan t an imal with an a ltered phenoty pe is identi fied, then i t can

be a ssu m ed th a t th e g en e th a t is m u ta ted is in v olv ed in r eg u la t in g th e pr ocess. Th is st r a teg y ,

applied in D. m ela no ga ster[5 4 5 6 ], has identified two genes inv olv ed in regu lating sleep Sh ,

encoding th e s h a k er K ch a nnel [5 4 ] an d the gen e encoding ex tra cellular GPI-an chored protein

ter m ed Sleepless [5 5 ] an d, wh en applied in C. elegans , ha s ident ified a cy clic-GMP-dependent

protein kinase [2 6 ,5 7 ].

Using curr ently av ai lable resources, howev er, part icularly in the fru i t fly , an inv est igator can

determ ine quickly w heth er the alt ered expression of genes identified in m icroarr ay studies will

affect sleep. In th e fru it f ly, th ere are lines av ailable with tra nsposable element s that disrupt gen e

funct ion by insertion in to identified genes [5 8 6 0]. Also av ailable is a librar y of RNA int erference

(RNAi) lines tha t ta rget 88% of protein-coding g enes [6 1 ]. T hese tra nsposon-insertion lines an d th e

+
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RNAi lines can lead to reduced gene fun ction. Th e pow er of av ailable D. melanoga stergen etic t ools is

tha t spatial a nd tem pora l control of gene expr ession ar e also possible using tw o- and th ree-

component tra nsgenic system s (for a rev iew, see [6 2 ]). Gene expression ca n be a ltered in both th e

positiv e and nega tiv e direction and can disregula te the gene of inter est identified in m icroarra y

studies. It is, therefore, feasible to test wheth er a g ene indentified in a m icroarra y experim ent a lso

regu lates sleep.

This technique was used in the study of the molecular ch aperone gene Hs pa5in the frui t f ly . The

expression of Hs pa5incr eases w ith sleep depriv ation in fru it f l ies [4 7 ] , mice [1 7 ], rats [1 3 ] and birds

[1 6 ]. Alth ough nu m erous stu dies indicate th at ex tended wa kefulness leads to increa sed expression

of the molecular ch aperone Hspa5, i t has been shown recently through genetic man ipulat ion of

Dros oph ilatha t altera tion ofHs pa5lev els does affect th e am oun t of recov ery sleep followin g sleep

depriv ation. Th ere is no alteration in th e am ount of baseline sleep or w akefulness. Incr eased

expression of Hs pa5increa ses the am oun t of sleep recov ery followin g sleep depriv ation [6 3 ]. By

contrast, in creased expression of a domina nt-negat iv e form of Hspa5decreases the am oun t of sleep

recov ery af ter s leep depriv ation [6 3 ]. Th ese observ ations are com patible with tw o possible

explanations. Hs pa5i tself could be a sleep-promoting m olecule w ith high er lev els ofHs pa5leading to

m ore recov ery sleep. Alternativ ely , a nd m ore l ikely , i s that higher lev els ofHs pa5delay act iv ation

of the UPR, as rev ealed by in vitro stu dies [6 4 ,6 5 ]. Hence, th ere is a n eed for m ore r ecov ery sleep,

the fina l m echan ism of defense. Thu s, processes contr olling the ba seline am oun ts of sleep v ersus

w a ke a n d th ose con tr olli n g r ec ov er y slee p fol low in g sleep de pr iv a tion ca n be sep a r a ted a t th e

m olecular lev el. The Hspa5 protein is inv olv ed in regu lating r ecov ery sleep. The role of Hspa5

protein, w hich w as identified by m icroarra y s as im porta nt to sleepwa ke fun ction, w as not

un derstood fully un til gene expression w as altered experim enta lly .

Microarrays and quantitative trait loci and the genetic basis of inter-individual differences

A spec ts of h u m a n sleep, su ch a s t im in g [6 6 ] and sleep dur ation [6 7 ], are heri table. Simi larly ,

m any sleep-related tra i ts are heri table in m ice [4 5 ,4 6 ,6 8 ] (for r ev iew, see [6 9 ,7 0 ]). Using m ouserecombina nt-inbred stra ins and a qua ntita tiv e trait locus (QTL) approach, a r egion on chrom osom e

1 3 inv olv ed in the r esponse to sleep depriv ation wa s identified [4 5 ]. QTL data w ere fur ther

cha ra cterized by considering g enes in the reg ion th at w ere expressed differentia lly dur ing sleep and

w a kefu ln ess i n m ic r oa r r a y st u di es [1 7 ,1 8 ]. By com bining haploty pe analy sis with data from gene-

expression profi ling by m icroarra y s, hu ndreds of genes located with in the QTL inter v al wer e

na rr owed dow n to a few g enes [7 1 ]. Howev er, only one of these genes, as determ ined by expression

profiling [i.e. Homer1a(a splice va rian t of the Hom er1 gen e)], has a differentia l incr ease in

expression w ith sleep depriv ation am ong t he recombin an t inbred strains used for QTL m apping

[1 8 ,7 1 ]. Moreover , there is a poly m orphism in the reg ula tory region of the Hom er1 gene that

probably impacts on th e tran script level observ ed in m icroarr ay prof il ing [7 1 ]. The prom oter

reg ion of the Hom er1 gen e contains m ultiple CRE sites that w ill bind the cy clic-AMP response

elem ent -bindin g pr otein (CREB) [7 2 ], and th e poly m orphism present in th is prom oter m ight

im pact CREB bindin g. Mice w ith low lev els of CREB ow ing to a deletion of the a nd isoform s of CREB

hav e reduced wakefulness during the early part of their n ight-t ime act iv e period [3 1 ] . Whether

this reduction in wa kefulness is m ediated, at least in par t, by reduced incr eases in Homer1awi th

w a kefu ln ess i n CREB h y pom or ph m ic e is cu r r en t ly u n kn ow n .

Thu s, microarray studies support th e hy pothesis that a v ariat ion in Homer1aexpression explains

differen ces in response to sleep depriv at ion [ 1 8 ,7 1 ].

Microarrays and molecular signatures of sleep deprivation and sleep disorders

Microarr ay s are a lso pow erful tools to identify the m olecular sign atu res of diseases (for rev iews, see

[7 3 ,7 4 ]). How ev er, they ar e only just beginning to be applied to the study of sleep depriv ation and
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sleep disord er s [7 5 7 8 ].

In D. melanoga ster, a m icroarr ay study identi fied that th e expression of the gene encoding a m y lase

increa ses with prolong ed wa kefulness [7 5 ] . In h um ans, am y lase increases in sa l iv a w ith s leep

depriv ation [7 5 ]. Th us, assessm ent of am y lase prov ides a potential bioma rker of sleep loss. Th e

im porta nce of ev alu ating w heth er th ere ar e biom ar kers for sleep hom eostasis (sleepiness) has been

em phasized prev iously [7 9 8 1 ]. Su ch biom ar kers w ill prov ide assessment s of sleepiness that could

be u sed in v a r iou s se tt in g s a n d pr ov id e a m ea n s for di st in g u ish in g in di v id u a ls w h o a r e pa r t ic u la r ly

sensitiv e to the effects of sleep depriv ation.

Molecular sign atu res m ight also be developed for sleep disorders, in par ticula r, t he comm on

condition known as obstruct iv e sleep apnea (for a rev iew, see [8 2 ]). In t his condition, br eath ing

stops repetitiv ely dur ing sleep, resulting in repeated interr uption of sleep and in cy clical repeated

hy poxic episodes [8 3 ]. Th ese hy poxic episodes result in free-radical production a nd a ctiv ation of the

tran scription factor NFB and inf lam m atory pathw ay s ( for rev iew, see [8 3 ]) . Microarr ay s hav e

shown t hat , in patients with obstru ctiv e sleep apnea, there ar e chan ges in tr anscript lev el in

sev eral gen es inv olv ed in m odulat ion of react iv e-oxy gen species (ROS), including h em e oxy gena se,

superoxide dismu tase and cat alase [7 7 ]. These chang es in obstru ctiv e sleep apnea pat ients are

suggestiv e of the activ ation of mech an ism s to m odulat e, and a dapt to, increased ROS developing inresponse to the frequent episodes of interm ittent hy poxia [7 7 ] . Thu s, tem poral ch anges in

m olecular -pathw ay components across the sleep period m ight provide a signat ur e of the presence of

th is an d perh aps oth er sleep disorder s [8 4 ].

Concluding remarks

Here, we h a v e a rg ued th a t m i croa rra y s a re a d iscov ery st ra teg y th a t h a s h a d a m a jor i m pa ct in

gener ating hy potheses about fun dam ental questions in sleep biology an d is beginnin g to identify

bi om a r ker s for both th e ef fects of slee p de pr iv a tion a n d for spec ific sleep di sor der s. Recen t da ta

suggest tha t exten ding w akefulness leads to ER stress an d sev eral different strategies, including the

use of specific phar m acological a gent s that m odify the pr ocess [8 5 ], support t his concept. Wh ether

sleep depriv ation leads to ER stress in the bra in in hu m an s is un known cur rent ly , althoug h it seem s

likely tha t th is w ill be the case because this has been dem onstra ted in all species stu died to date.

Giv en th at ER stress indu ced by sleep depriv ation is also foun d in th e liv er, i t is conceiv able th at ER

stress might be dem onstrated in peripheral leukocytes in hu m ans an area for fur ther study .

The concept of modulation of sy na ptic plasticity with sleep or wak e state, ar ising from m icroarra y

studies, is also supported by inv estigation of phosphory lation of the g luta m ate r eceptors

calcium /calm odulin-dependent pr otein kin ase II (CaMKII) an d gly cogen sy nth ase kinase 3 (GSK3 )

[8 6 ]. Howev er, other approaches, at least in dev eloping an im als [8 7 ], suggest th at strength ening of

syn aptic connections occur s dur ing sleep, not wa kefulness, wh ich is com patible with t he

im provem ents in perform an ce of specific tasks that occur in h um an s from before to after sleep (for

rev iew, see [8 8 ]). Thu s, furt her study of this concept is required because the situa tion m ight be

m ore complex. It is conceiv able tha t different n eur ona l groups respond differently with respect to

sleep or w ake cha ng es in syn aptic plasticity .

The concept that sleep is a stage of macr om olecular syn thesis is relativ ely recent, a lthough

compatible with ea rlier data th at protein sy nth esis occur s dur ing sleep [8 9 ,9 0]. Furth er studies to

v a li da te th is a r e r equ ir ed . In pa r t ic u la r , it w il l be im por ta n t to det er m in e th e m ole cu la r ba sis for

the switch from energ y resour ces being used during w akefulness to support n eur ona l f iring to those

be in g u sed du r in g slee p for sy n th esis of k ey m ole cu le s.

A lth ou g h m u ch h a s bee n a ccom pl ish ed , m u ch m or e r em a in s t o be don e (Box 2 ). Microarr ay studies

of chan ges in gene expression du ring the cy cle of sleep and wa kefulness in th e nem atode and

zebrafish should fur ther c larify w hat funct ions of sleep ar e conserv ed across phy logeny . Sleep and
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w a kefu ln ess, u n lik e th e cir ca di a n clock, w h ic h ca n fu n ct ion a s a cel l-au ton om ou s m olec u la r

nega tiv e-feedback loop, are contr olled by intera cting n eur onal circuits (for rev iew, see

[5 3 ,9 1 9 3 ]). Thu s, understan ding sleep and wa kefulness at th e level of tra nscription r equir es the

study of cha ng es in gen e expression w ithin identified populat ions of neuronal cells. Identification

an d isolation of particula r n eur ona l cell populat ions would th en be the basis of a m icroarra y -based

exam ination of gene expr ession in m odel org an ism s. This is possible with laser m icrodissection,

high-throughput in situhy bridization or the u se of flow cy tometr y for cell separa tion (for a rev iew of

th ese m eth ods, see [9 4 ]). The first attem pt to assess cha ng es in th e tran script level in a specific

neur onal population was m ade by Maret et al. , w ho used microarr ay s to study RNA isolated from

neu rons expressing Homer1agene [1 8 ]. In th is experim ent, a poly -A binding protein (PAPB) w as

expressed in tra nsgenic m ice under t he control of the promoter of the Hom er1 gene, follow ed by the

pur ification of the PAPB an d the bound m RNA. T his stu dy identified sev eral tr an scripts w ith

cha ng ing expression in Hom er1a-expressing neu rons after sleep loss, inc luding , a s predicted, th e

tra nscript of the Homer1agene [1 8 ].

Box 2. Future applications of microarrays in sleep research

To identify conserv ed gene ex pression cha nges in sleepw ake periods in a dditiona l m odel

org an ism s to identify key conserv ed sleep fun ctions.

To determ ine ch an ges in gen e expression du ring sleepw ake periods and on sleep

depriv ation in identified neuronal populations.

To determ ine cha ng es in gen e expression du ring sleepw ake an d sleep depriv ation in

peripheral org an s; separat ing effects of sleepwa ke an d sleep depriv ation from clock

influences.

To dev elop m olecular sign atu res for diag nostic applications and for identify ing

indiv iduals part icular ly at risk of adv erse out comes stemm ing from sleep depriv ation

an d sleep disorders.

Recent a dv an ces in sequen cing t echn ologies, such a s short-read high -thr oug hpu t sequen cing (RNA-Seq), m ake i t c lear tha t cur rent m ethods, including m icroarr ay s, ar e not identi fy ing a l l of the

tra nscriptiona l landscape of m am m alian cells [9 5 ]. RNA-Seq detects as ma ny as 25 % m ore gene

tran scripts than m icroarr ay s. Th us, high-through put sequencing of the tra nscriptom e during sleep

an d wak efuln ess will prov ide additional in form ation concer ning cha ng es in gen e expression

be tw een th ese be h a v ior a l st a tes.

A lth ou g h th e con tr ol of sleep a n d w a kefu ln ess or ig in a tes in th e br a in a n d m a n y fu n ct ion s of sleep

ar e centered on t he br ain, lack of sleep affects peripheral orga ns, as seen by the effects of sleep

depriv ation on m etabolism [9 6 ] and cardiac health [9 7 ]. Ther efore, micr oar ray studies also need to

extend t o periphera l organ s so that the effect of sleep and w akefulness, as w ell as sleep depriv ationon gen e expression in hear t, lun g an d kidney , am ong other s, can be assessed.

In fur ther inv estigat ions in different species, different n eur onal gr oups an d different organ s, study

design s need to be used to separa te th e effects of sleep or w ake from circ adia n clock influ ences, as ha s

be en don e in r ec en t stu di es [1 8 ].

One of the g oals of clinica l resear ch is to identify th e ear liest possible stage of progressiv e diseases, so

tha t treat m ent can be initiated. Thu s, alth oug h th e search es for predictiv e biom ar kers of sleep

disorders and th e deleterious effects of sleep depriv ation ha v e only just begun , m icroarra y -based

strategies ar e already beginnin g to identify path wa y s to provide both diagn ostic inform ation about

th e presence of a specific sleep disorder a nd pr ognostic inform at ion.

Acknowledgements

We a r e g r a tefu l to Da n ie l Bar r et t a n d Jen n ifer Mon toy a for th ei r h el p in pr ep a r a tion of t h is
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m anu script . The original research w as supported by NIH gran ts HL60287 , AG1 7 62 8 and

HL66 61 1 , an d by the NIH/NHGRI Ruth L. Kirch stein Postdoctoral Fellow ship HG003 96 8 to K.R.S.

Footnotes

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Article information

Trends Mol Med. Author manuscript; available in PMC 2010 September 20.

Published in final ed ited form as:

Trends Mol Med. 2009 February; 15(2): 7987.

Published online 2009 January 21. doi: 10.1016/j.molmed.2008.12.002

PMCID: PMC2942088

NIHMSID: NIHMS230888

Miroslaw Mackiewicz, John E. Zimmerman, Keith R. Shockley, Gary A. Churchill, a ndAllan I. Pack

Center for Sleep an d Respiratory Neurob iology, University of Pennsylvania School of Med icine, Philadelphia, PA 19104, USA

Division of Sleep Med icine/Department of Medicine, University of Pennsylvania School of Medicine, Philade lphia, PA 19104, USA

The Jackson Labor atory, Bar Harbo r, ME 04609, USA

Corresponding author: Pack, A.I. (Email: pack/at/mail.med.upenn.edu)

Copyright noticeand Disclaimer

Publisher's Disclaimer

The publishe r's final edited version of this article is available at Trends Mol Med

See other a rticles in PMC that citethe published article.

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