Homer1a Is a Core Brain Molecular Correlate of Sleep Loss

Homer1a is a core brain molecular correlate of sleep loss

Ste´ phanie Maret*, Ste´ phane Dorsaz*, Laure Gurcel*, Sylvain Pradervand†, Brice Petit*, Corinne Pﬁster*, Otto Hagenbuchle†, Bruce F. O’Hara‡, Paul Franken*, and Mehdi Tafti*§

*Center for Integrative Genomics and †Lausanne DNA Array Facility, University of Lausanne, Ge´nopode, CH-1015 Lausanne, Switzerland; and ‡Department of Biology, University of Kentucky, Lexington, KY 40506-0225

Communicated by Michael Rosbash, Brandeis University, Waltham, MA, October 24, 2007 (received for review October 15, 2007) Sleep is regulated by a homeostatic process that determines its need takes these interacting factors into account. We show that short- and by a circadian process that determines its timing. By using sleep term sleep loss induces changes in brain gene expression for a few deprivation and transcriptome profiling in inbred mouse strains, we genes only. These genes are all part of a highly specific pathway show that genetic background affects susceptibility to sleep loss at involved in neuronal protection and recovery after waking-induced the transcriptional level in a tissue-dependent manner. In the brain, glutamate overstimulation. Homer1a expression best reflects the response to sleep loss. Time- course gene expression analysis suggests that 2,032 brain transcripts Results are under circadian control. However, only 391 remain rhythmic when We have previously reported (7) that the dynamics of sleep need mice are sleep-deprived at four time points around the clock, sug- varies strongly among inbred mouse strains, with AKR/J (AK) mice gesting that most diurnal changes in gene transcription are, in fact, showing a dramatic increase in delta power after 6-h sleep depri- sleep–wake-dependent. By generating a transgenic mouse line, we vation, whereas DBA/2J (D2) mice have a blunted response. show that in Homer1-expressing cells specifically, apart from Through quantitative linkage analysis, a significant quantitative Homer1a, three other activity-induced genes (Ptgs2, Jph3, and Nptx2) trait locus (QTL) was identified on mouse chromosome 13 (Dps1: are overexpressed after sleep loss. All four genes play a role in delta power in slow-wave sleep 1) that explains Ͼ50% of variance recovery from glutamate-induced neuronal hyperactivity. The consis- in delta power after sleep deprivation in BXD recombinant inbred tent activation of Homer1a suggests a role for sleep in intracellular lines (RIs) derived from inbred mouse strains C57BL/6J (B6) and calcium homeostasis for protecting and recovering from the neuronal D2 (7). The best polymorphic marker associated was D13Mit126 at activation imposed by wakefulness. 46.5 cM (95% CI ϭ 25 cM), suggesting a large QTL region (Ϸ38 Mb). However, based on the most recent high-resolution, single- homeostasis ͉ microarray ͉ mRNA tagging ͉ sleep deprivation ͉ nucleotide polymorphism (SNP) genetic map of BXD RIs (10) sleep function (http://gscan.well.ox.ac.uk/gs/strains.cgi), the smallest differential region corresponds to an 11-Mb sequence flanked by SNPs wo main processes regulate sleep. A homeostatic process rs3669221 and rs397202. According to the Ensemble database (Mus Tregulates sleep need and intensity according to the time musculus release 46), this region contains 33 known genes, 15 spent awake or asleep. A circadian process regulates the appro- potential unknown coding sequences, and three pseudogenes. priate timing of sleep and wakefulness across the 24-h day. A Among these, the short splice variant of the Homer1 (Homer1a) highly reliable index of the homeostatic process is provided by gene is the only transcript in the region that was previously reported the amplitude and prevalence of delta (1- to 4-Hz) oscillations to be among the up-regulated genes after sleep deprivation (11–14). in the electroencephalogram (EEG) of nonrapid eye movement However, the specificity of this finding, compared with other gene (NREM) sleep (hereafter, ‘‘delta power’’). Delta power is high expression changes after sleep loss, has not yet been established. at sleep onset and decreases during sleep, in parallel with sleep To confirm and verify whether differences in sleep need between depth. Sleep deprivations and naps induce a predictable increase mouse strains are regulated at the transcriptional level, mice of or decrease, respectively, in delta power during subsequent three genotypes (AK, B6, and D2) were deprived of sleep by gentle sleep. The interaction between homeostatic and circadian pro- handling [see supporting information (SI) Materials and Methods] cesses is mathematically described in the two-process model of for 6 h, starting at light onset, and killed at the same time of day sleep regulation, which provides a framework for prediction and [Zeitgeber time (ZT)6] with their non-sleep-deprived controls. interpretation of a large body of experimental data (1). Because it is believed that sleep fulfills a brain-specific function, we Among hypotheses concerning the physiological function of also sampled the liver as a peripheral reference organ. Microarray waking-induced changes in sleep, the most compelling suggests that results were analyzed by a two-way ANOVA, with strain and sleep plays a key role in synaptic plasticity (2, 3). More specifically, condition as main factors. Venn diagrams summarizing the data are EEG delta power during NREM sleep has been shown to play a shown in Fig. 1 A and B and suggest that very few genes show critical role in learning-induced plasticity (4–6). In general, the consistent changes in expression across genetic backgrounds. We prediction is that local neural activation due to specific behavioral use the terms ‘‘consistent’’ and ‘‘reliable’’ hereafter only for tran- (cognitive) demands imposes a burden on the brain which neces- sitates sleep and which is reflected by the EEG delta power. On the basis of mathematical modeling and experimental data, Author contributions: S.D. and L.G. contributed equally to this work; O.H., P.F., and M.T. designed research; S.M., S.D., L.G., B.P., C.P., O.H., and B.F.O. performed research; S.M., S.D., we have shown that sleep need, as indexed by the EEG delta power, L.G., S.P., B.F.O., P.F., and M.T. analyzed data; and S.M., S.D., L.G., S.P., P.F., and M.T. wrote is under genetic control (7), which is of direct relevance for the paper explaining the interindividual vulnerability to sleep loss in human The authors declare no conflict of interest. subjects (8, 9). However, deciphering the molecular bases of sleep Data deposition: The data reported in this paper have been deposited in the Gene need is rendered difficult because the contributions of the homeo- Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE9444). static and circadian processes are difficult to separate and because §To whom correspondence should be addressed. E-mail: [email protected]. the impact of genetic background on brain gene expression is poorly This article contains supporting information online at www.pnas.org/cgi/content/full/ understood. From a series of gene-profiling experiments, we here 0710131104/DC1. report a comprehensive transcriptome analysis that specifically © 2007 by The National Academy of Sciences of the USA

20090–20095 ͉ PNAS ͉ December 11, 2007 ͉ vol. 104 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710131104 Downloaded by guest on September 26, 2021 part of a general stress–response pathway induced by enforced wakefulness in most organs, and, therefore, is not specific to the brain. The most significant decrease was found for the cold-induced RNA binding protein (Cirbp) in both tissues, again suggesting a general rather than a brain-specific implication (SI Tables 1 and 2). In brain, the expression of Homer1a was most affected by both genotype and sleep loss. In situ hybridization analysis confirmed the strong induction of Homer1a transcript after sleep deprivation and indicated a restricted up-regulation in cortex, striatum, and hippocampus (Fig. 1C). Homer1 encodes several transcripts by alternative splicing, among which long forms are constitutively expressed while two short forms, Homer1a and Ania-3, are activity-induced. These postsynpatic density proteins bind calcium-signaling mole- cules and have been implicated in synaptic plasticity. In contrast to most other activity-induced genes, Homer1a overexpression seemed highly specific. For instance, brain-derived neurotrophic factor (Bdnf), a plasticity-related gene, showed a significant increase in AK and D2 only; the immediate early gene Fos did not show a significant increase in D2; and Arc, Egr1, and Egr3 were induced by sleep deprivation mainly in AK (SI Table 1). Thus, comparisons among genotypes, and between brain and liver, identified Homer1a as the most specific transcriptional index of the whole brain in response to sleep loss. We then analyzed the time course of Homer1a induction by real-time quantitative RT-PCR in a dose–response experiment. Mice of all three strains were sleep-deprived for 1, 3, and6hand killed together with their time-matched controls. An additional group, which was also sleep-deprived for 6 h, was killed2hinto recovery sleep. As shown in Fig. 1D, Homer1a is rapidly and strongly induced by sleep deprivation in a dose-dependent manner. Parallel to this increase, Homer1a expression decreases with in- creasing accumulated sleep in non-sleep-deprived animals, and its relative level remains significantly higher than in the time-matched controls after only2hofrecovery sleep, indicating that the time course of Homer1a expression closely parallels sleep need. How- Fig. 1. Transcriptome analysis of the brain and liver after sleep deprivation ever, the fold change of Homer1a expression after 6-h sleep NEUROSCIENCE in three inbred mouse strains indicates that Homer 1a is speciﬁcally upregu- lated in the brain. (A and B) Venn diagrams of a two-way ANOVA microarray deprivation was similar between D2 and AK, which represent the data analysis of total RNA extracted from brain (A) and liver (B) in AK, B6, and two extreme strains in their response to sleep deprivation, and was D2 inbred mouse strains at ZT6 after a 6-h sleep deprivation. Results are significantly higher than in B6 mice (Fig. 1E). In accord with our reported as a function of genetic background (genotype), experimental con- findings, sequence comparisons in public databases for the Homer1 dition (sleep deprivation), and their interaction. (C) Homer1a is overexpressed region indicated that D2 and AK share the same SNP haplotype, after sleep deprivation (SD) in the cortex and caudate putamen (Left) and in which is different from that of B6, although no coding sequence the hippocampus (Right). (D) Mean (Ϯ1 SEM) forebrain mRNA levels of differences between any of the three strains can be identified. This Homer1a for AK, B6, and D2 mice after SDs of 1, 3, and 6 h (pink bars), with finding suggested that, even though Homer1a is the best candidate their time-matched controls (gray bars). All SDs started at light onset (ZT0). for the Dps1 QTL segregating with response to sleep loss between Effects of recovery sleep on expression were assessed by allowing2hof recovery after 6-h SD (ZT8). Homer1a expression was affected by SD, geno- D2 and B6 mice, other genes may affect sleep need in other inbred type, and time of day (P Ͻ 0.05; three-way ANOVA). (E) Fold change increase strains, or posttranscriptional or translational changes of Homer1a of Homer1a expression level after6hofSDvaries among strains. Ratios may differ between D2 and AK mice. between SD and control mRNA levels at ZT 6 were calculated as described in SI Materials and Methods, with their standard errors. The effect of SD on Time-of-Day Effects of Sleep Loss on Brain Transcriptional Changes. As Homer1a expression is statistically different between B6 and the two other shown in Fig. 1D, the level of Homer1a expression under baseline strains (ϩ, P Ͻ 0.006; two-way ANOVA). conditions shows significant variation that might be either due to a direct circadian effect or driven by the diurnal sleep–wake distribution, as we suggest. To separate time-of-day and homeostatic scripts either increased or decreased in all three inbred strains and effects on brain gene expression, we have simulated the time course across experimental conditions. of sleep need (quantified as EEG delta power) according to Sleep deprivation induced changes in only 42 brain probe sets in published assumptions and parameters (7) under entrained base- B6, 92 in D2, and 188 in AK, among which 52 sets were affected by line conditions and after 6-h sleep deprivation at four time points genotype. To our surprise, sleep loss induced almost three times around the 24-h day. If a gene is implicated in the processes more transcriptional changes in liver compared with whole brain underlying sleep need, then changes in its expression can be (Fig. 1A). As in brain, among all transcripts with significant changes expected to parallel the predicted time course of sleep need (Fig. in liver, Ϸ50% changed as a function of genetic background. Thus, 2A). Under control conditions, sleep need is high at sleep onset, for genes differentially expressed in each tissue of the three strains which coincides with light onset (ZT0), and during the latter part in response to sleep deprivation, we have focused on those showing of the active period (ZT18), whereas sleep need is lowest between a significant two-way ANOVA interaction (see SI Tables 1 and 2). ZT6 and ZT12 (i.e., dark onset) (Fig. 2A). Under sleep deprivation As reported in refs. 11, 15, and 16, sleep deprivation most signifi- conditions, these pronounced sleep–wake-dependent changes are cantly up-regulated the expression of heat shock protein genes in expected to be strongly reduced. Thus, we sleep-deprived mice of both brain and liver, strongly suggesting that this group of genes is the three strains for 6 h, starting at ZT0, ZT6, ZT12, or ZT18, and

Maret et al. PNAS ͉ December 11, 2007 ͉ vol. 104 ͉ no. 50 ͉ 20091 Downloaded by guest on September 26, 2021 Fig. 2. Around-the-clock transcriptome analysis of the brain after sleep deprivation A B AK B6 D2 indicates that most changes are affected by ’erusserp peelS‘ ’erusserp ZT 0 6 12 18 0 6 12 18 0 6 12 18 behavioral states. (A) Simulation of sleep need in the around-the-clock sleep- Homer1a control deprivation experiment. Sleep-need dynam- C Cycling ics in the control (gray) were modeled Control SD in SD mathematically according to Franken et al. AK B6 D2 AK B6 D2 AK B6 D2 (7): lights on at ZT0, lights off at ZT12. The ZT 6 ZT 6 increase in sleep-need during the four 6-h ZT [h] ZT12 sleep deprivations (SD) was determined by assuming a saturating exponential increase Control SD during wakefulness (red dashed lines). The D ZT18 ZT0 ZT0 red solid line connects values of sleep need ZT 0 reached at the end of the SDs. (B and C)SD f feoc feoc ZT18 ZT6 ZT18 ZT6 ZT 6 affects the cycling pattern of gene expression in the brain. Rhythmic transcripts were se- s

oc lected, as outlined in SI Materials and Meth- ods, and their temporal expression patterns ZT12 ZT12 were aligned according to phase. (B) Enlarge- sin coeff ment of heat map for Homer1a gene in control condition. For AK, B6, and D2, the four E time points (ZT0, 6, 12, and 18) are repre- Homer1a Bmal1 sented in triplicate. Green and red represent ZT12 minimal and maximal expression levels, respectively. (C Left) The 2,032 genes cycling in min max controls are depicted. The peak time of expression is indicated at left. (Center) The

ZT18 same genes are represented after SD. Note )X001( that the rhythmic accumulation of most tran- Egr3 Per2 scripts is severely blunted after SD. (Right) langiS ANRm langiS The 391 probe sets for which cycling expression proﬁles are not affected by SD (with the peak of expression indicated at right). (D) Cycling genes shown in C are plotted according to their amplitude and phase. Homer1a, Arc Dbp outlined in green, is the most rhythmic transcript under control conditions. (E) Temporal expression proﬁles of representative homeostatic (Left) and circadian (Right) genes in ZT 0 whole brain. Normalized expression signals from microarray experiments are plotted as a function of time for control (solid lines) and ZT [h] SD (dashed lines) mice of each strain. Each ZT 6 point is the mean Ϯ SEM of three pools of three animals.

killed them together with their home-cage controls (n ϭ 9 per strain as Homer1a, Arc, and Egr3 show a high amplitude variation (up to per time per condition; i.e., total 216). A linear statistical model was 6-fold for Homer1a), closely following the diurnal sleep–wake first used (see SI Materials and Methods) and identified 2,540 probe distribution, and no, or greatly reduced, variation after sleep sets that were significantly changed at any time point (false-positive deprivation. Although the pattern of expression of circadian genes rate; false discovery rate Ͻ5%). These probe sets were then remains little affected by sleep deprivation, their relative levels can assessed for time-of-day variation separately in the baseline and be significantly affected (unchanged for Bmal1, increased for Per2, sleep-deprivation conditions. Under the baseline condition, Ϸ8% and decreased for Dbp;Fig.2E), as we also reported in refs. 17 (2,032; see SI Table 3) of probe sets detected in the brain showed and 18. a significant time-of-day pattern of expression (Fig. 2 C and D), with Comparison of sleep-deprived and control mice at all four time points again revealed that Ͻ2% (343 probe sets) of the expressed two major, opposite phases of peak expression (between ZT6 and Ͼ ZT12 and between ZT18 and ZT0, respectively; Fig. 2D). Under the genes in the brain are up-regulated ( 70%, or 249 probe sets) or down-regulated (Ͻ30%, or 94 probe sets) by sleep loss (SI Table 5). sleep-deprivation condition, only 391 (SI Table 4) of the 2,032 sets Significant interaction between condition and time of day was at baseline were still significantly affected by time of day, indicating detected for 585 probe sets. Again, the most significantly overex- that most others changed according to the sleep–wake distribution, pressed gene after sleep deprivation was Homer1a, followed by rather than as a result of (or in addition to) a direct circadian effect. those belonging to stress–response and synaptic-plasticity gene Among the 391 transcripts that maintained a significant cycling groups. The major functional gene groups that reduced their pattern after sleep deprivation, a large majority reached maximum expression after sleep deprivation concern protein synthesis, mem- levels of expression between ZT0 and ZT6 and between ZT12 and brane trafficking, and protein transport (SI Table 5) (12). Real-time ZT18 (Fig. 2 C and D). Among all rhythmic transcripts under quantitative RT-PCR verification for 41 candidate genes (found control conditions, Homer1a clearly showed the largest amplitude here and by others) at ZT6 confirmed our microarray findings at of variation (Fig. 2 B, D, and E), beyond that of all canonical this reference time point (SI Table 6). circadian genes. Time course of expression of six representative genes under the two conditions is depicted in Fig. 2E. As predicted Cell-Specific Transcriptional Changes Due To Sleep Loss. Previous by the simulation analysis (Fig. 2A), activity-regulated genes such studies (12, 19, 20) investigated transcriptomes of different brain

20092 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710131104 Maret et al. Downloaded by guest on September 26, 2021 structures, rather than the whole brain. In contrast to most other A ~100kb organs, the brain is a highly heterogeneous tissue, with specialized H1a Ania-3 H1b/c regions and nuclei having very different functional roles. Whole- 1 234Exons5 678910 brain transcriptome, therefore, suffers several potential limitations, including dilution of low copy-number transcripts and missing those 5’ 6’ with opposing patterns of transcriptional changes among brain regions. On the other hand transcriptome analysis of selected B 1234 5 regions also has drawbacks because of our limited knowledge of BAC exactly where functionally significant molecular changes can be PABP IRES eGFP Flag expected and because, even in well defined regions, cell types vary ZEO greatly. To overcome these limitations, we chose to analyze changes Recombination in mRNA profiles of those neurons that are selectively and specif- 179Kb 36Kb g a BAC l PABP IRES eGFP

ically activated by sleep deprivation. To this end, we used a modified F ZEO mRNA tagging technique originally established in Caenorhabditis Flip-out of Zeo elegans and Drosophila (21–23). BAC PABP IRES eGFP

We have shown here that Homer1a is the transcript most Flag consistently increased by enforced wakefulness. The Homer1 gene BXD-F2 pronucleus injection is therefore a good marker for neuronal populations activated by 22 g

Chrom. la PABP IRES eGFP

sleep loss but not restricted to a single structure. We thus generated F BAC-based transgenic mice by replacing the first five exons of 11 33 Homer1 (corresponding to activity-induced Homer1a transcript; +- +- +-Tg mice Tg mice Control Fig. 3A) by a FLAG-tagged poly(A) binding protein 1 (PABP) C 700 D 500 Sleep Deprived -+-+ followed by internal ribosome entry site (IRES)-eGFP (Fig. 3B). 300 142 Because PABP binds poly(A) tails of mRNAs, affinity-purification Loading control of FLAG-tagged PABP proteins from whole-brain lysates is ex- FLAG PABP eGFP 98 pected to coprecipitate all mRNAs from neurons expressing Flag Homer1a. Seven transgenic lines were obtained, and analysis indi- 64 36 cated that at least three lines expressed the transgene at very similar eGFP amounts, at both mRNA and protein levels (Fig. 3 C and D). Also, 22 the induction of the transgene by 6-h sleep deprivation was very Fig. 3. A transgenic mouse model to analyze neuron-specific gene expres- similar to that of endogenous Homer1a, indicating that our BAC sion. (A) Schematic representation of the genomic structure of Homer1. The construction contains the regulatory elements for the correct putative transcriptional initiation site is depicted by a bent arrow at the functional expression of Homer1a (Fig. 3D). beginning of exon 1; the translational stops for short activity-induced The expression of this construct was also verified by in situ Homer1a (H1a) and Ania-3, as well as for long constitutively expressed hybridization (Fig. 4A). Although eGFP inserted after the IRES did Homer1b/c (H1b/c) are indicated by black circles. Intron 5 is here divided into not result in reliable signal under epifluorescence, riboprobes four segments (4.4 kb of Homer1a 3Ј UTR, 5.7 kb up to Ania-3, 1.4 kb of NEUROSCIENCE against Homer1a and eGFP clearly indicated that both are similarly Ania-3-specific sequence, and 18.8 kb to exon 6). The Homer1a-specific exon coexpressed in the same brain regions (Fig. 4A). Double fluores- 5Ј extends exon 5 by the intron 5 sequence. The Ania-3-specific exon 6Ј sits cent in situ hybridization (FISH) with riboprobes against Homer1a within intron 5 (adapted from ref. 25). (B) General strategy for generating Homer1a-PABP transgenic mice. The relative position of the Homer1a gene in and eGFP revealed that, at least in two transgenic lines, eGFP was the fully sequenced 227,644-bp RP23–262I3 BAC clone (GenBank accession no. almost exclusively expressed in Homer1a-expressing neurons (Fig. AC120347), and the different steps used to introduce a PCR-amplified con- 4B), which represented Ϸ40% of the neurons in the cingulate cortex struction containing PABP cDNA, followed by IRES-eGFP and the zeocine (ZEO) and 30% in the dorsal striatum (not counted in the hippocampus). selection cassette flanked by FRT sites (hashed boxes) by BAC recombination All mRNA immunoprecipitations and microarray data presented in the EL250 bacterial stain (see SI Materials and Methods). (C) Transgenic mice below are from a single transgenic line (line 36). Sleep recordings (Tg) were identified by RT-PCR with the primer pairs depicted in B for the in Homer1-PABP transgenic mice indicated that they react to sleep presence of the FLAG (primers 1/1), PABP (primers 2/2), and eGFP (primers 3/3). deprivation similar to their wild-type littermates (SI Fig. 5). The (D) Western blot verification of transgene expression in transgenic mice line specificity of this mRNA pull-down method was also verified by 36 (Tg) indicated the presence of the FLAG and eGFP and the up-regulation of the FLAG after sleep deprivation. The loading control is a nonspecific band quantitative RT-PCR with probes specific for eGFP, FLAG, generated by the GFP antibody. Homer1a, and Hcrt (a gene expressed only in the lateral hypothal- amus, where Homer1a expression is very low). The results indicated that eGFP, FLAG, and the endogenous Homer1a were enriched were identified (SI Table 7), among which the most significant and induced by sleep deprivation, whereas only trace amounts of ones—Homer1a, Egr2 (NGFI-B), and Fosl2 (Fos-like antigen 2)— Hcrt could be detected (SI Fig. 6). were similarly induced after sleep loss in the pull-down and the Immunoprecipitated mRNAs were prepared from Homer1- whole-brain extracts, suggesting that gene expression changes in PABP transgenic mice with (n ϭ 6) or without (n ϭ 6) a 6-h sleep the pull-down samples recapitulate the most significant changes at deprivation at light onset for gene expression profiling. For com- the whole-brain level. In addition, several unique immediate-early parison, RNA extractions were also made from supernatants (n ϭ genes were specifically identified in pull-down samples that might 4) after immunoprecipitation and from transgenic whole brains be coinduced with Homer1a, namely prostaglandin–endoperoxide (n ϭ 8). To test for transcriptional changes after sleep deprivation synthase 2 (Ptgs2), junctophilin 3 (Jph3), and neuronal pentraxin 2 in Homer1-expressing cells, we proceeded in two steps: (i)we (Nptx2). Interestingly, both Jph3 and Nptx2 are activity-induced identified probe sets enriched in the pull-down extracts and (ii) through either ryanodine receptor-mediated intracellular calcium among those probe sets, we compared sleep deprivation with mobilization (Jph3) or activity-induced AMPA receptor synaptic control condition in both pull-down (6 vs. 6-chip comparison) and clustering (Nptx2). Among the very few down-regulated transcripts whole-brain (4 vs. 4-chip comparison) extracts. We found that 4,728 (SI Table 7), we have identified another activity-induced gene, probe sets were significantly enriched at 5% false discovery rate 4-nitrophenylphosphatase domain and nonneuronal SNAP25-like when pull-downs were compared with both supernatant and whole- protein homolog 1 (Nipsnap1), suggesting that plasticity genes can brain extracts (SI Materials and Methods). Again, very few genes be up- or down-regulated by sleep deprivation.

Maret et al. PNAS ͉ December 11, 2007 ͉ vol. 104 ͉ no. 50 ͉ 20093 Downloaded by guest on September 26, 2021 Nipsnap1, one of the proposed plasticity genes (24), is actually Homer1a eGFP A B Antisense Sense down-regulated after sleep deprivation in our mRNA tagging Cg IPA experiment. Three different genes in mammals encode Homer proteins. D esnesitnA Homer1 encodes constitutively expressed long-form proteins, CPu whereas short-form Homer1a is activity-induced (25). Homer1

a1remoH long-form proteins dimerize and interact with metabotropic gluta- Pir mate receptors and increase calcium from intracellular stores. Short-form proteins, which lack the dimerization domain, function as natural activity-dependent dominant negative forms that regulate the scaffolding and signaling capabilities of the long forms and PFG reduce glutamate-induced intracellular calcium release (26, 27). We have recorded sleep, and the response to a 6-h sleep deprivation, in esneS Homer1 (all forms) mutant mice but found a very similar pattern

d compared with their wild-type littermates (data not shown). This e

gr finding could be expected due to the fact that, because Homer1a e functions as a dominant negative form of the long forms, consti- M 5µm tutive loss of all isoforms might not result in any specific sleep phenotype. Fig. 4. Colocalization of Homer 1a and FLAG-tagged PABP eGFP transcripts. Conceptually, spontaneous or enforced wakefulness repre- (A) In situ hybridization with Homer1a and eGFP antisense riboprobes indicated that both are expressed in similar brain structures. Cg, cingulate cortex; sents a stressor activating a series of stress–response mecha- CPu, caudate putamen; Pir, piriform cortex. (B) Confocal images of FISH nisms of the organism, which, at the transcriptional level, could analysis. FISH experiments were performed with both Homer1a (red) and eGFP be translated into the induction of genes such as heat shock (green) riboprobes at the same time and revealed that almost all positive proteins in most tissues. However, unlike in other organs, neurons were double-labeled, indicating the colocalization of the endoge- brain-specific stress–response pathways are primarily triggered nous Homer1a and the transgene. Negative control conditions were obtained by glutamate. Glutamate is the major excitatory neurotrans- with both sense riboprobes. mitter in the central nervous system and acts through either ionotropic or metabotropic receptors (mGluRs). Long Hom- er1 tetramers bind group I mGluRs and inositol 1,4,5- Discussion triphosphate receptors, thus enabling efficient calcium release The results presented here demonstrate that6hofsleepdepriva- from intracellular stores, whereas monomeric Homer1a com- tion, which importantly impacts sleep physiology and behavior, petitively disrupts synaptic glutamatergic signaling complexes results in only minimal changes in brain transcriptional adaptation. to reduce glutamate-induced intracellular calcium release (26, As reported for changes in delta power (7), we also showed here 27). Homer1 also activates ryanodine receptors and L-type that sleep loss-induced transcriptional changes are largely affected calcium channels (28, 29). Interestingly, Jph3, which was by genetic background. As opposed to other studies that did not identified by our mRNA-tagging strategy as being up- take genetic background into account and did not contrast their regulated by sleep deprivation, has been shown to play a major findings to peripheral tissue (12, 15, 20), our results indicate that role in ryanodine receptor-mediated, calcium-induced open- only a few genes reliably change expression after sleep deprivation. ing of small-conductance, calcium-activated potassium (SK) The surprising finding that sleep loss induced a larger number of channels (28, 30). SK channels are responsible for the gener- changes in liver than in brain suggests either that sleep deprivation ation of slow afterhyperpolarizations in neurons of the nucleus might have a specific impact on the liver or that the brain might be reticularis thalami and thus contribute to the EEG slow waves protected against major transcriptional changes. characteristic of NREM sleep (31). Another important aspect of the present study is the interaction According to Tononi and Cirelli (3), plastic processes occurring between the homeostatic and circadian processes. Although we did during wakefulness result in increased synaptic strength, whereas not sleep-deprive the animals under constant conditions, and the role of sleep is to downscale synaptic strength to a basal level. therefore the direct and indirect effects of light, for instance, on Homer1a transcription is rapidly up-regulated in neurons in re- gene expression could not be accounted for, we have shown that a sponse to synaptic activity induced by long-term potentiation, large majority (Ͼ80%) of changes in gene expression were driven seizure, inflammation, stimulant drugs, or even selectively in the by the prior sleep–wake history. hippocampus of rodents by exploratory behavior (32, 33). In this We also adopted, and further developed, a reliable mRNA context, Homer1a, by buffering intracellular calcium and disassem- tagging technique to investigate gene expression changes in neu- bling synaptic glutamatergic signaling complexes, could play a rons. This technique can be used to evaluate different neuronal pivotal role in synaptic downscaling. Because Homer1a and the subpopulations without the burden of sampling several structures other genes identified here (Egr2, Fosl2, Ptgs2, Jph3, and Nptx2) are or using labor-intensive laser microdissection to isolate neurons. all induced by stressful conditions such as seizure, stroke and The results of this technique confirmed that sleep loss-induced hypoxia, and inflammation, an alternative, complementary view transcriptional changes occur for very few genes, among which could be that they play a primary brain-protective or recovery role. Homer1a remains the most specific. This view is also of relevance for the etiology of neuropsychiatric In addition to Homer1a, we identified overexpression of other disorders because it is increasingly recognized that stress is impli- genes involved in synaptic plasticity, but only Egr2 and Homer1a cated in many of such disorders (34). Both environmental and were found to consistently change across experiments. Others pharmacological stressors up-regulate Homer1a mRNA in key reported overexpression for a number of plasticity-related genes, structures involved in higher brain functions (35): the same struc- and these observations are commonly used in support of a func- tures in which Homer1a is up-regulated after sleep deprivation. It tional role for sleep in plasticity. Because the expression of most has also been shown that overexpression of Homer1a after inflam- plasticity-related genes was not reliably changed, our findings do not mation, seizure, and psychostimulant or antipsychotic drug use support such a general conclusion and instead suggest that the plays a major role in neuroprotection (28, 35). It is tempting to molecular mechanisms might not be identical for most plasticity- relate the dramatic improvement in depression in humans after regulated genes. In this context it is important to note that sleep-deprivation (36) to the sleep deprivation-induced up-

20094 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710131104 Maret et al. Downloaded by guest on September 26, 2021 regulation of Homer1a. In conclusion, converging evidence strongly lated by using the bicinchoninic acid assay (Pierce) with BSA as a implicates Homer1a as a brain-coping marker against stressors, and standard. Eighty micrograms of each fraction were analyzed by our findings suggest that Homer1a might represent the molecular SDS/PAGE, followed by Western blotting using antibodies as link between sleep, cognition, and neuropsychiatric disorders. follows: mouse anti-tubulin 1/1,000 (Santa Cruz), goat anti- Homer1a 1/200 (Santa Cruz), mouse anti-Flag M2 1/300 (affinity- Materials and Methods purified; Sigma), and rabbit anti-GFP 1/2,500 (AbCam). Secondary Animal Handling. All experiments were performed in accor- antibodies were all coupled with HRP, except for the anti-goat dance with the protocols approved by the Ethical Committee antibody, which was IRDye800-conjugated for Lycor analysis. of the State of Vaud Veterinary Office, Switzerland. Sleep- In situ hybridizations with coronal cryosections of 12 ␮m were deprivation and sleep-recording procedures are described in SI performed according to Allen Brain Atlas protocols (enzymatic Materials and Methods. BCIP/NBT revelation) (37). All reagents and solutions were pur- chased and prepared based on Eurexpress II in situ hybridization cRNA Preparation, cDNA Microarray Hybridization, and Real-Time consortium instructions. GFP and Homer1a riboprobes were syn- RT-PCR. For the first experiment, we isolated total RNA from whole thesized by in vitro transcription on a linearized pGEM-Easy vector brain and liver by using the RNAXEL kit (Eurobio), treated the (Promega) containing the corresponding sequences. The cDNA RNA with DNase, and cleaned it using RNeasy columns (Qiagen). insert of this plasmid was generated by RT-PCR from mouse brain Equal quantities of total RNA from three individual mice of each RNA, using the following primers: Homer1a forward, 5Ј- strain were pooled in triplicate (nine mice of each strain in each GCTGTCAGAAGCTTAGGATGTG-3Ј; Homer1a reverse, condition). A hybridization mixture containing 15 ␮g of biotin- 5Ј-AAAGTGCAGAAAGTCCAGCAGC-3Ј; GFP forward, 5Ј- ylated cRNA was hybridized to GeneChip Mouse Expression Set GAGCTGGACGGCGACGTAAACG-3Ј; and GFP reverse, 5Ј- 430. Chips were washed, scanned, and analyzed with Affymetrix AGGACCATGTGATCGCGCTTCTC-3Ј. GeneSpring software. FISH was performed as described in ref. 38, using anti-DIG- For the around-the-clock microarray experiment, RNA from POD 1/600 (Roche), anti-FLU-AP 1/100 (Roche), and SA-Alexa whole brain was isolated and purified with the RNeasy Lipid Tissue 488 (Molecular Probes) and counterstained with DAPI (Sigma). Midi kit (Quiagen) and DNase-treated. All RNA quantities were assessed with a NanoDrop ND-1000 spectrophotometer, and the Transgenic and mRNA Tagging. Transgenic mice were generated as quality of RNA was controlled on Agilent 2100 bioanalyzer chips. described in Fig. 3. See SI Materials and Methods for details. Equal amounts of total RNA were pooled from three mice within each of the 24 experimental groups (three strains, two conditions, We thank K. Harshman, A. Paillusson, and M. Bueno for assistance in 4ZTϭ 24; in triplicate: 24 ⅐ 3 ϭ 72 chips). Three micrograms of microarray and real-time RT-PCR analyses at the Lausanne DNA Array each of these pools were used to perform the chip array experiment, Facility; P. Descombes, M. Docquier, D. Chollet, and C. Delucinge for according to the Affymetrix Gene Expression procedure. Twelve assistance in microarray and real-time RT-PCR analyses at the Geneva micrograms of biotinylated cRNA from each sample were frag- Genomics Platform, National Center for Competence in Research Frontiers mented and hybridized to GeneChip Mouse 430 2.0 arrays, accord- in Genetics; S. Excoffier for help in the transgenic construction; P. Seeburg, M. Schwarz (Max Planck Institute, Heidelberg, Germany), and P. Worley ing to standard procedures. Microarray analyses and qPCR verifi- (Johns Hopkins School of Medicine, Baltimore, MD) for providing Homer1 cations were performed as reported in SI Materials and Methods. mutant mice; and A. Vassali for constructive discussions. This work was NEUROSCIENCE supported by the Swiss National Science Foundation and the State of Vaud Immunoblot and in Situ Hybridization. Total protein extract was (M.T.) and in part by National Institutes of Mental Health Grant MH67752 prepared with RIPA lysis buffer. Protein concentration was calcu- (to P.F.).

1. Dijk DJ, Franken P (2005) in Principles and Practice of Sleep Medicine, eds 21. Roy PJ, Stuart JM, Lund J, Kim SK (2002) Nature 418:975–979. Kryeger TH, Roth TH, Dement W (Saunders, Philadelphia), pp 418–434. 22. Kunitomo H, Uesugi H, Kohara Y, Iino Y (2005) Genome Biol 6:R17. 2. Krueger JM, Obal F (1993) J Sleep Res 2:63–69. 23. Yang Z, Edenberg HJ, Davis RL (2005) Nucleic Acids Res 33:e148. 3. Tononi G, Cirelli C (2006) Sleep Med Rev 10:49–62. 24. Satoh K, Takeuchi M, Oda Y, Deguchi-Tawarada M, Sakamoto Y, Matsubara 4. Huber R, Ghilardi MF, Massimini M, Ferrarelli F, Riedner BA, Peterson MJ, K, Nagasu T, Takai Y (2002) Genes Cells 7:187–197. Tononi G (2006) Nat Neurosci 9:1169–1176. 25. Bottai D, Guzowski JF, Schwarz MK, Kang SH, Xiao B, Lanahan A, Worley 5. Marshall L, Helgadottir H, Molle M, Born J (2006) Nature 444:610–613. PF, Seeburg PH (2002) J Neurosci 22:167–175. 6. Molle M, Marshall L, Gais S, Born J (2004) Proc Natl Acad Sci USA 26. Worley PF, Zeng W, Huang G, Kim JY, Shin DM, Kim MS, Yuan JP, Kiselyov 101:13963–13968. K, Muallem S (2007) Cell Calcium 42:363–371. 7. Franken P, Chollet D, Tafti M (2001) J Neurosci 21:2610–2621. 27. Xiao B, Tu JC, Worley PF (2000) Curr Opin Neurobiol 10:370–374. 8. Van Dongen HP, Baynard MD, Maislin G, Dinges DF (2004) Sleep 27:423–433. 28. Kakizawa S, Kishimoto Y, Hashimoto K, Miyazaki T, Furutani K, Shimizu H, 9. Tucker AM, Dinges DF, Van Dongen HP (2007) J Sleep Res 16:170–180. Fukaya M, Nishi M, Sakagami H, Ikeda A, et al. (2007) EMBO J 26:1924–1933. 10. Shifman S, Bell JT, Copley RR, Taylor MS, Williams RW, Mott R, Flint J 29. Yamamoto K, Sakagami Y, Sugiura S, Inokuchi K, Shimohama S, Kato N (2006) PLoS Biol 4:e395. (2005) Eur J Neurosci 22:1338–1348. 11. Cirelli C, Gutierrez CM, Tononi G (2004) Neuron 41:35–43. 30. Moriguchi S, Nishi M, Komazaki S, Sakagami H, Miyazaki T, Masumiya H, 12. Mackiewicz M, Shockley KR, Romer MA, Galante RJ, Zimmerman JE, Saito SY, Watanabe M, Kondo H, Yawo H, et al. (2006) Proc Natl Acad Sci USA Naidoo N, Baldwin DA, Jensen ST, Churchill GA, Pack A (2007) Physiol 103:10811–10816. Genomics 31:441–457. 31. Blethyn KL, Hughes SW, Toth TI, Cope DW, Crunelli V (2006) J Neurosci 13. Nelson SE, Duricka DL, Campbell K, Churchill L, Krueger JM (2004) Neurosci 26:2474–2486. Lett 367:105–108. 32. Tappe A, Klugmann M, Luo C, Hirlinger D, Agarwal N, Benrath J, Ehren- 14. Huber R, Tononi G, Cirelli C (2007) Sleep 30:129–139. gruber MU, During MJ, Kuner R (2006) Nat Med 12:677–681. 15. Terao A, Steininger TL, Hyder K, Apte-Deshpande A, Ding J, Rishipathak D, 33. Kato A, Ozawa F, Saitoh Y, Fukazawa Y, Sugiyama H, Inokuchi K (1998) J Biol Davis RW, Heller HC, Kilduff TS (2003) Neuroscience 116:187–200. Chem 273:23969–23975. 16. Wisor JP, Morairty SR, Huynh NT, Steininger TL, Kilduff TS (2006) Neuroscience 34. Southwick SM, Vythilingam M, Charney DS (2005) Annu Rev Clin Psychol 141:371–378. 1:255–291. 17. Franken P, Thomason R, Heller HC, O’Hara BF (2007) BMC Neurosci 8:87. 35. Szumlinski KK, Kalivas PW, Worley PF (2006) Curr Opin Neurobiol 16:251– 18. Wisor JP, O’Hara BF, Terao A, Selby CP, Kilduff TS, Sancar A, Edgar DM, 257. Franken P (2002) BMC Neurosci 3:20. 36. Giedke H, Schwarzler F (2002) Sleep Med Rev 6:361–377. 19. Cirelli C, Faraguna U, Tononi G (2006) J Neurochem 98:1632–1645. 37. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, 20. Terao A, Greco MA, Davis RW, Heller HC, Kilduff TS (2003) Neuroscience Boguski MS, Brockway KS, Byrnes EJ, et al. (2007) Nature 445:168–176. 120:1115–1124. 38. Ishii T, Omura M, Mombaerts P (2004) J Neurocytol 33:657–669.

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