Disruption of CREB Function in Brain Leads to Neurodegeneration

article

Disruption of CREB function in brain leads to neurodegeneration

Theo Mantamadiotis1,3*, Thomas Lemberger1*, Susanne C. Bleckmann1, Heidrun Kern1, Oliver Kretz1, Ana Martin Villalba1, François Tronche1, Christoph Kellendonk1, Daniel Gau1, Josef Kapfhammer2, Christiane Otto1, Wolfgang Schmid1 & Günther Schütz1 *These authors contributed equally to this work.

Published online: 22 April 2002, DOI: 10.1038/ng882

Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To deﬁne its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem–/– background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.

Introduction by phosphorylation and bind as homo- or heterodimers to the Waves of neuronal apoptosis are crucial for shaping the complex cAMP response element (CRE) in the promoters of their target architecture of the developing brain, whereas progressive, genes3. CREB has long been implicated in neuronal function,

© http://genetics.nature.com Group 2002 Nature Publishing regional loss of neurons underlies the irreversible pathogenesis of with much recent interest centered around its role in the mainte- various neurodegenerative diseases in adult brain. During devel- nance of long-term memory4. More recently, several studies opment, the decision between neuronal survival or death is involving overexpression of dominant-negative CREB suggested determined by access to neurotrophic support and activation of a role for CREB as a survival factor in various cellular models5−9, excitatory neurotransmitter receptors. Both events activate signal possibly acting downstream of the Akt/PKB survival pathway10. transduction pathways that eventually protect neurons from We have used genetic disruption to study the function of the apoptotic cell death. In the case of chronic neurological diseases, CREB family members. We previously generated two different various triggering mechanisms, including metabolic impair- Creb1 mutations in the mouse. The first, Creb1α∆, led to the ment, excitotoxicity, free radical production or biochemical generation of a hypomorphic mutation11,12, whereas the sec- abnormalities resulting from genetic mutations, have been iden- ond, Creb1–/–, is a true null mutation that results in perinatal tiﬁed as causative agents in neuronal death1. However, the mole- death of homozygous mice13. Determining the significance of cular pathways involved in the transduction of these signals into complete CREB loss in adult mice thus required the generation cell death are largely unknown. The mechanisms responsible for of conditional Creb1 mutant mice. One additional complicat- neuronal apoptosis in neurodegenerative diseases may at least ing factor is that in both Creb1α∆ and Creb1–/– mutants, Crem partially overlap with those that are involved in development. expression is upregulated in many organs, including brain12,13. Anti-apoptotic signals exert their effect on cell survival The functional importance of CREM upregulation has until through various mechanisms, including modulation of gene now remained unknown. Here we report the generation of expression. Neurotrophin- and activity-dependent gene expres- mice with a conditional mutation of Creb1 (Creb1loxP). We use sion is mediated by several neuronal signal transduction cas- these mice to show the result of disrupting both Creb1 and cades, such as the PI3K/Akt pathway, the MAPK pathway, the Crem in brain either during development or postnatally. These Ca2+/CaMK and cAMP/PKA pathways, that converge on the studies show that CREB and CREM regulate similar neuronal CREB family of leucine-zipper transcription factors2. This tran- survival processes, as only neurons devoid of both CREB and scription factor family is comprised of CREB, the related proteins CREM undergo apoptosis. Moreover, our genetic dissection cAMP response element modulatory protein (CREM) and the reveals an unexpected difference between developmental and activating transcription factor 1 (ATF1). All three are activated postnatal roles of CREB and CREM. Specifically, when both

1Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. 2Institute of Anatomy, University of Basel, Switzerland. 3Present address: Peter MacCallum Cancer Institute, St Andrews Place, East Melbourne, 3002, Australia. Correspondence should be addressed to G.S. (e-mail: [email protected]).

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Fig. 1 Generation of mice deficient for CREB in the nervous system. a a, Organization of Creb1 encompassing exons 9–10. Exon 10 was flanked with loxP sites in two steps: First, we generated the modified allele by homologous recombination in ES cells. Second, tran- sient expression of Cre recombinase resulted in removal of the selection cassette (neomycin resistance and thymidine kinase), gen- erating Creb1loxP and Creb null alleles. A scheme depicting the wildtype gene locus, targeting vector and resulting alleles are shown. Triangles represent loxP, black rectangles represent exons; white rectangles represent probes used for Southern-blot analysis and arrows represent primers used for PCR genotyping32. H, HindIII; K, KpnI; X, XbaI. b–q, Expression of Cre recombinase in the cre transgenic lines and pattern of recombination in mutants. Nescre transgenics express Cre throughout the developing brain by E12 (data not shown). Cre expression becomes restricted to the proliferating ventricular zones by E16.5, as revealed by Cre immunohistochemistry (b). At E18, CREB is expressed throughout the brain in b c Creb1loxP/loxP mice (c) but lost in Creb1NesCre brains (d). Camkcre4 d transgenics show high Cre expression in all cortical layers (e), in the entire hippocampus (h), in amygdala (k) and striatum (l). Cre-mediated recombination is detected by comparison of Creb protein levels in Creb1loxP/loxP control animals at 12 wk (f,i,m) to those of Creb1Camkcre4 mutant littermates (g,j,n). Recombination affects all cortical layers equally (compare f with g) and is extensive in hip- e f g pocampus (compare i with j) and striatum (compare m with n). D1cre transgenics show Cre expression restricted to the striatum (o) correlating with CREB loss in Creb1D1cre mutants (q) compared with Creb1loxP/loxP controls (p). Original magnification: ×10 (b–d,o), ×30 (e–l,p,q) and ×400 (m,n,inserts). h i j

neuronal and glial lineages (Fig. 1b). CREB immunoreactivity in Creb1Nescre mice was lost in most of the developing brain by embryonic day (E) 12.5 k l m n (data not shown). By E18.5, Creb1Nescre mice showed extensive loss of CREB in brain (Fig. 1d). The high CREM upregulation observed in these mice (data not shown) suggested a compensatory role for CREM in the absence of CREB. To circumvent this possible compen- o p q sation, Creb1Nescre mice were further crossed to Crem–/–

© http://genetics.nature.com Group 2002 Nature Publishing mice15, yielding Creb1NescreCrem–/– double-mutant mice, which lacked both Creb and Crem in brain. At four weeks, all genotypes except Creb1Nescre Crem–/– mutants were represented, suggesting embry- onal or perinatal death of mice lacking both CREB and CREM in brain. By analyzing litters at E18.5 and newborn (P0) mice, we found that Creb1Nescre Creb1 and Crem are inactivated in neuronal and glial precur- Crem–/–mice were present at roughly the expected mendelian sors during development, generalized cell death occurs in the ratio. Upon closer examination, we saw that newborn nervous system. By contrast, postnatal disruption of both Creb1NescreCrem–/– mutants invariably did not suckle, as there CREB and CREM function leads to progressive neurodegener- was no evidence of milk in their stomachs, and, as a result, they ation that is restricted to the dorsolateral striatum and to the died within one day after birth. CA1 and dentate gyrus regions of the hippocampus. Brain development in Creb1NescreCrem–/– mice Results All brain structures were present in Creb1NescreCrem–/– brains at Generation of mice lacking both Creb and Crem in brain E16.5, E18.5 and P0, indicating that CREB and CREM do not Mice either hypomorphic for CREB11,12 or completely lacking have major roles in brain formation. Histological analysis CREB13 or CREM14,15 show normal embryonic brain develop- showed a reduced cell density throughout the brain at E18.5 ment. In Creb1α∆ and Creb1–/– mutant mice, reduction or loss of and P0, accompanied by an increase in cells with pyknotic CREB is accompanied by upregulation of CREM12,13. As all three nuclei and enlarged ventricles (Fig. 2). Notably, these dramatic members of the CREB family can potentially compensate for one changes were more extensive in P0 brains, indicating an accu- another, it is notable that only CREB and CREM, but not ATF1, mulation or acceleration of cell death over this time period. In are detectable in wildtype mouse neurons (data not shown). the highly affected cortical areas of mutant P0 brains, the corti- As an initial step toward obtaining mice devoid of CREB in cal plate was completely disorganized and only a few pyramidal the nervous system, we generated Creb1loxP/loxP mice by homol- cells could be found within loosely arranged clusters of small ogous recombination in embryonic stem (ES) cells (Fig. 1a). round cells (Fig. 2d). Instead of a dense layer of large pyramidal Creb1loxP/loxP mice showed normal expression of CREB in all neurons, only sparsely distributed, small round cells were present tissues, including brain (Fig. 1c). These mice were crossed to in the pyramidal cell layer of areas CA1 and CA3 of the hip- mice harboring a nestin-driven Cre recombinase transgene16, pocampus (Fig. 2f). In other areas, such as the thalamus or hypo- to generate Nescre Creb1loxP/loxP (Creb1Nescre) mice. The nestin thalamus, the cytoarchitecture was better preserved and speciﬁc promotor induces Cre expression early, before separation of nuclei could be identiﬁed. The number of differentiated neurons

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Fig. 2 Cellular integrity is compromised in brains of mice lacking both Creb and Crem. a,b, Histological analysis of Creb1NescreCrem+/– control (a) and a b Creb1NescreCrem–/– (b) brain sections at P0. c–h, Comparing Nissl stainings reveals extensive loss of large neurons and an increased number of cells with pyknotic nuclei, especially in the cortical plate (c,d) and in the hippocampal area CA1 (e,f) of mutant mice. The thalamus or hypothalamus were also affected (g,h), but to a lesser degree. i,j, In the olfactory bulb, the mitral cell layer was markedly reduced. v, ventricular space. Original magniﬁcation: ×10 (a,b), ×200 (c–h), ×400 (i,j). Scale bar, 500 µm (a,b); 20 µm (c–h); 10 µm (i,j).

in these nuclei was reduced, however, and many neurons were smaller than those of control tissue (Fig. 2g,h). In the olfactory bulb of Creb1NescreCrem–/– brains, the mitral cell layer showed the c d greatest cell loss (Fig. 2j). Similar changes were seen in rhinal and limbic cortical areas (data not shown).

Severe neuronal loss during brain development due to apoptosis To examine whether the reduced number of neurons was a result e f of increased cellular degeneration, reduced proliferation or both, we labeled cells with Ki-67 and TUNEL and stained for activated caspase 3 in mutant and control brains (Fig. 3). Cell proliferation, as assessed by Ki-67 labeling, revealed no obvious differences between Creb1NescreCrem–/– double-mutant (Fig. 3c) and Creb1loxP/loxPCrem+/– or Creb1NescreCrem+/– control brains g h (Fig. 3a,b) of mice at E18.5, indicating that proliferation of neurons in the ventricular zones is largely unaffected by the absence of CREB and CREM. In contrast, apoptosis occurred, to varying extents, in all areas of the brain (data not shown). Cells positive for TUNEL and activated caspase 3 were abundant and typically found in the cortical plate, within layers V and VI of i j Creb1NescreCrem–/– brains (Fig. 3f,i). Apoptotic cells were rare and present only in the subventricular zone of cerebral cortex of control mice (Fig. 3d,e,g,h). Apart from the Creb1NescreCrem–/– brain,

© http://genetics.nature.com Group 2002 Nature Publishing other genotypes, including Creb1Nescre or Crem–/– (data not shown) and Creb1NescreCrem+/– (Fig. 3e,h), showed control levels of apoptosis, further supporting the link between neuronal death and the loss of both CREB and CREM.

In brain of Creb1NescreCrem–/– mice at E16.5, TUNEL- positive cells were already apparent, but restricted to ear- a b c lier developing areas, such as the amygdala and small regions of the thalamus (Fig. 4a,b). Areas that develop later, such as the cerebral cortex, the hippocampus and caudate putamen, showed little or no labeling for apoptotic cells at E16.5; however, the number of cells undergoing apoptosis was significantly increased by E18.5 and P0 (Fig. 4a,b). We also observed TUNEL labeling in the trigeminal ganglion and dorsal-root ganglia (data not d e f shown), indicating that the observed increase in apoptotic death was not confined to the central nervous system. The mechanism by which loss of CREB leads to neuronal death is unknown, but may involve pro-survival factors such as Bcl2, which is a putative CREB target gene6,17,18. We therefore carried out an analysis of factors involved in regulation of cellular survival by RNase g h i Fig. 3 Normal proliferation, but increased neuronal death, in mice lacking both CREB and CREM in brain. a–c, The proliferative activity of the ventricular zones of Creb1loxP/loxPCrem+/– and Creb1NescreCrem+/– control mice (a and b, respectively) was similar to that of Creb1NescreCrem–/– animals (c). Scattered cell death was seen throughout the control brains, but was greatly increased in Creb1NescreCrem–/– brains. d–i, Compared with control mice (d,e,g,h), cells in layers VI and V of the mutants show intense TUNEL labeling (f) and activated caspase 3 staining (i). Original magnification: ×200.

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a b

Fig. 4 Neuronal degeneration is initiated between E16.5 and E18.5 in the absence of CREB and CREM. a,b, Regional and developmental onset of cell death in NescreCreb1loxP/+Crem+/– controls (a) and Creb1NescreCrem–/– double-mutants (b). Detection of apoptotic cells by TUNEL labeling of 7-µm parafﬁn sections during different developmental stages is shown. Cells of the cortical plate and the caudate putamen show little staining at E16.5, but the number of positively stained cells is markedly increased in double-mutant animals at E18.5 and at P0. In the reticular thalamic nuclei and the amygdala, which develop early, apoptotic cells are already apparent in double-mutant embryos at E16.5. This results in a high cell loss at later stages. Magniﬁcation: ×400.

protection assay. We found no difference in the mRNA levels of Progressive post-natal neurodegeneration both pro- and anti-apoptotic factors Bcl2, Bcl2l, Bcl2l2, Bak1, As Creb1NescreCrem–/– mice lose CREB during early neuronal devel- Bax and Bad (see Web Fig. A online). Loss of expression of anti- opment and die perinatally, we were only able to study these mice

© http://genetics.nature.com Group 2002 Nature Publishing apoptotic Bcl2 family members is thus unlikely to be responsible until birth, when neurons are still differentiating. To determine the for the neuronal apoptosis seen in our model. consequences of CREB loss in postnatal neurons, we crossed the Creb1loxP/loxP mice with transgenic mice (Camkcre4) expressing Cre recombinase postnatally, under the control of the 8.5-kb promoter a b fragment of the calcium/calmodulin-dependent protein kinase II- α gene (Camk2a). We found high levels of Cre expression in striatum, nucleus accumbens (part of the basal ganglia), thalamus, amygdala, cortex and the hippocampus of the Camkcre4 transgenic mice (Fig. 1e,h,k,l). In Camkcre4 Creb1loxP/loxP mutants (Creb1Camkcre4), Cre-mediated recombination was extensive in all areas of the brain where the recombinase is expressed (Fig. 1, compare panels f,i,m with panels g,j,n). CREB was not detectable in pyramidal cells of hippocampal CA1, CA2, and CA3 (Fig. 1j) or in granule cells of the dentate gyrus, except for a thin strip of cells on the internal face. Extensive recombination was also observed in striatum (Fig. 1n), amygdala (data not shown) and in all cortical c d layers (Fig. 1g) as indicated by the absence of detectable CREB. The functional compensation by Crem as seen in Creb1Nescre mutants prompted us to generate Creb1Camkcre4Crem–/– double-mutant mice. Offspring were viable, and genotyping revealed the expected mendelian ratios. Notably, we saw a neurological phenotype in older (more than six months) Creb1Camkcre4Crem–/– double- mutants. When suspended by their tail for more than 20 seconds, e f Fig. 5 Neurological and morphological phenotype of Creb1Camkcre4Crem–/– double-mutants. a,b, Creb1Camkcre4Crem+/– control animals at 12 mo have normal limb reflexes when suspended by the tail (a), whereas Creb1Camkcre4Crem–/– double-mutants show an abnormal feet-clasping behavior (b). c–f, Dark-field, low-power magnification (×10) of brain sections from Creb1Camkcre4Crem+/– controls (c,e) and Creb1Camkcre4Crem–/– double-mutants (d,f) at 12 mo. Note the shrinkage of the striatum (compare c with d) and the marked atrophy of the hippocampus in the double-mutants (compare e with f).

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a b c d e

f g h i j

k l m n o

Fig. 6 The striatum and the hippocampus from Creb1Camkcre4Crem–/– double-mutants undergo progressive neurodegeneration. a–o, Cre (a–e) and neuN (f–o) immunostaining of brain sections from Creb1Camkcre4Crem+/– controls at 1.5 mo (a,f,k) and Creb1Camkcre4Crem–/– double-mutants at 1.5 mo (b,g,l), 2 mo (c,h,m), 3 mo (d,i,n) and 6 mo (e,j,o). Striatal degeneration is progressive (b–e) and is confined to the dorsolateral area. The nucleus accumbens is still intact at the age of 24 wk (e). In hippocampus, neuronal loss accumulates in CA1 and dentate gyrus while CA3 remains intact (g–j). Panels k–o show high-power magnification of the CA1 region. Magnification: ×25 (a–j), ×400 (k–o).

double-mutant animals retracted their hind limbs toward their copy of Crem showed no signs of gliosis, demonstrating the abil-

© http://genetics.nature.com Group 2002 Nature Publishing trunks in a dystonic fashion, rather than extending them as did ity of CREM to compensate for the loss of Creb (Fig. 7a,c). control littermates (Fig. 5a,b). This feet-clasping phenotype is As predicted by the massive wave of apoptosis observed in observed in several mouse mutants with neurological impairment Creb1NescreCrem–/– mutants, staining of Creb1Camkcre4Crem–/– due to neurodegeneration19−23. At the morphological level, the brain sections with the nuclear Hoechst 33342 dye revealed brain of the Creb1Camkcre4Crem–/– double-mutants showed consid- many apoptotic cells with highly condensed and fragmented erable atrophy of the striatum and the hippocampus (Fig. 5c−f). nuclei in the regions of neurodegeneration (see Web Fig. B To determine whether the atrophy of striatum and hippocam- online). Similarly, staining for the activated form of caspase 3 pus was caused by a wave of cell death at a particular stage of revealed scattered positive cells in CA1, dentate gyrus and dor- development or by progressive cell loss, we analyzed the time- sal striatum, whereas no positive cells could be detected in course of neuronal loss in brain of Creb1Camkcre4Crem–/– double- control animals (see Web Fig. B online). Similar to what we mutants. At 1.5 months, neuronal loss, as visualized by neuN or observed in the Creb1NescreCrem–/– mutants, we could not Cre immunostaining, is modest in hippocampus and almost detect significant differences in Bcl2 immunostaining between undetectable in striatum (Fig. 6, compare panels a,f,k with panels control and Creb1Camkcre4Crem–/– double-mutant mice (see b,g,l). The lesion becomes progressively larger with time (2, 3 and Web Fig. A online). 6 months) in both structures, eventually leading to extensive atrophy of the dorsolateral striatum, a marked reduction in Genetic dissection of striatal degeneration thickness of dentate gyrus and a complete elimination of CA1 We next sought to verify the pattern of striatal degeneration in an (Fig. 6e,j,o). Thus, the hippocampal and striatal lesions are the independent mouse line in which disruption of Creb1 was more result of the accumulation of scattered neuronal loss during a restricted to the striatum. We therefore used the D1cre transgenic progressive neurodegenerative process. Striatal degeneration was line, in which Cre expression is directed by the dopamine recep- restricted to the dorsolateral region and did not affect the tor D1A gene (Drd1a) promoter. To achieve faithful expression in nucleus accumbens. The CA1 region of the hippocampus this transgenic model, we used a YAC of 140 kb containing the showed the most severe cell loss, whereas degeneration in the entire Drd1a as an expression vector for Cre recombinase. dentate gyrus was milder and the CA3 region was unaffected. Expression of the transgene recapitulated the expression pattern The speciﬁcity of the lesion was revealed by GFAP immunostain- of the endogenous Drd1a. Speciﬁcally, we found high expression ing, which indicates the areas where astrogliosis has been levels of Cre in striatum, nucleus accumbens and olfactory tuber- induced by neuronal injury. We detected massive astrogliosis in cles (Fig. 1o). We also found weaker expression in layer VI of the the dorsal striatum, CA1 and dentate gyrus of cortex, CA2 in hippocampus and some nuclei of the thalamus Creb1Camkcre4Crem–/– double-mutants (Fig. 7b,d−f). Fainter glio- (data not shown). Recombination, as shown by CREB immuno- sis was observed in amygdala, cortex and thalamus (data not cytochemistry, overlapped with regions of Cre expression in shown). By contrast, control animals carrying a single functional Creb1D1cre mutants (Fig. 1p,q).

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Fig. 7 Astrogliosis in striatum and hippocampus. a–f, GFAP immunostaining a b reveals no signs of gliosis in Creb1Camkcre4Crem+/– control mice, which retain one functional copy of Crem (a,c). By contrast, massive astrogliosis is visible in striatum (b) and hippocampus (d) of Creb1Camkcre4Crem–/– double-mutants at 12 wk. In the hippocampus of double mutants, gliosis is restricted to CA1 and dentate gyrus (dg, e). High-power magniﬁcation of GFAP staining in dorsolateral striatum (f). Magniﬁcation: ×10 (a–d), ×30 (e), ×400 (f).

c d had no impact on neuronal survival or survival of the mice. The same holds true for brains from Crem–/– mice, which show no defect in neuronal survival. As mice devoid of both CREB and CREM in the brain during development show a severe loss of neurons (Fig. 2), we conclude that CREM upregulation in Creb1 mutant mice is sufficient to maintain cellular survival. Decreased cell density in Creb1NescreCrem–/– brain resulted from the increased incidence of neuronal apoptosis and not from e f decreased proliferative activity (Figs 3 and 4). The first evidence of apoptosis occurred at E16.5, in the earlier-developing areas, such as the amygdala and reticular thalamic nuclei (Fig. 4). Later- developing areas, such as the cerebral cortex, hippocampus and caudate putamen, showed little evidence of apoptosis at E16.5; however, the number of cells undergoing apoptosis was significantly increased by E18.5 and P0 (Fig. 4). Increased apoptotic nuclei were also seen in the trigeminal ganglion and dorsal-root ganglia (data not shown), indicating that the observed increase The phenotype of Creb1D1CreCrem–/– double-mutants was most in apoptotic death was not confined to the central nervous sys- striking in 7-month-old mice. As seen with the tem. As significant CREB loss occurs as early as E12.5, it is Creb1Camkcre4Crem–/– double-mutants, degeneration specifically unlikely that CREB influences neuronal survival at periods affected the dorsolateral striatum of Creb1D1creCrem–/– double between E12.5 and E16.5. However, we can not rule out the pos- mutants (Fig. 8a,b). Notably, not only was the spatial specificity of sibility that small amounts of residual CREB may be sufficient to the striatal degeneration retained in the Creb1D1creCrem–/– strain, support survival during this time period. but the kinetics were also virtually identical to that observed in The selective loss of neurons in the Creb1Camkcre4Crem–/– and Creb1Camkcre4Crem–/– double-mutants (Fig. 8c−f). These observa- Creb1D1creCrem–/– mice also supports a role for CREB and CREM tions suggest that the pattern of degeneration in striatum is depen- in adult neuronal survival. However, the specificity of neuronal loss

© http://genetics.nature.com Group 2002 Nature Publishing dent on the intrinsic sensitivity of striatal neurons. observed in the postnatal forebrain contrasts with the earlier generalized effects observed in Creb1NescreCrem–/– mutants. Thus, devel- Discussion opmental and postnatal functions of CREB and CREM are only This study demonstrates that CREB family members are crucial partially overlapping. We hypothesize that loss of CREB in differen- in neuronal survival in vivo. Absence of CREB and CREM in tiated neurons reduces the probability of neuronal survival rather developing brain results in generalized cell death, whereas post- than being an absolute requirement for survival, as observed in the natal disruption of transcription mediated by CREB or CREM developing brain. In both postnatal models presented here, the sen- triggers selective and progressive neurodegeneration. Loss of sitive areas degenerate progressively because of scattered apoptotic only CREB in brain, either during development or postnatally, cell death, mimicking the typical ‘one-hit’ dynamics of cell loss described for several neurodegenerative disorders24. It is possible that differential a b survival in the postnatal brain is modu- lated by both region-speciﬁc expression of protective factors and the network properties of the vulnerable structures. The dynamics of degeneration is then likely to be determined by intrinsic properties of the affected areas, such as CA1, dentate gyrus and striatum, which may explain why the time-courses of striatal degeneration are so similar in the two c d e f independent postnatal models presented

Fig. 8 Striatal neurodegeneration in Creb1D1creCrem–/–mice. a,b, At 7 mo, immunostaining for Cre in Creb1D1creCrem+/– control animals (a) and Creb1D1creCrem–/– double-mutants (b) reveals a clear lesion in the dorsolateral striatum. c–f, Cre immunostaining showing the evo- lution of the lesion in Creb1D1creCrem–/– mice at 1.5 mo (c), 2 mo (d), 3 mo (e) and 7 mo (f). Mag- niﬁcation: ×10 (a,b), ×25 (c–f).

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Nescre here. If the effect of CREB loss is to lower the threshold of survival, Creb1 mutant mice are subfertile, to the point that it was impractical it is possible that death may be triggered by a particular pattern of to use this genotype for breeding. In each case, the expected number of Creb1NescreCrem–/– mice was 1 in 8. Similarly, to generate mice with postna- neuronal electrical activity. In conjunction with the role of CREB in 4 tal Creb loss (see Camkcre4 and D1cre transgenic lines below), we used the synaptic plasticity , these observations open up the possibility that following mating schemes to obtain double-mutants: (i) Camkcre4 CREB may control processes commonly involved in both apoptosis Creb1loxP/loxPCrem+/– males × Creb1loxP/loxPCrem–/– females (1 in 4 proba- and synaptic plasticity. bility of obtaining a double mutant) and (ii) D1creCreb1loxP/+Crem+/– Hippocampus and striatum are particularly vulnerable to males × Creb1loxP/loxPCrem–/– females (1 in 8 probability of obtaining a insult in several contexts, including neurometabolic disorders, double-mutant). Mice were bred in a genetic background comprising a seizure, ischemia episodes and neurodegenerative diseases such mix of 129SvOla, C57/BL6 and FVB/N. We genotyped mice with the loxP 32 as Huntington disease. Several recent studies have shown that Creb1 allele by PCR (Fig. 1a) as previously described and detected mutated forms of huntingtin, ataxin-1, atrophin-1 and the cre transgenes by dot-blot analysis of tail DNA. The Crem null allele was detected by PCR using primers designed within the Crem allele and androgen receptor proteins, all of which carry an expanded lacZ cassette. polyglutamine stretch and cause neurodegenerative diseases, interact and sequester CREB-binding protein (CBP) and 25–28 cre transgenic lines. To generate the Camkcre4 transgenic line, we cloned TAF2C1 transcriptional co-factors . These interactions are the open reading frame (ORF) encoding Cre recombinase, fused to a thought to deplete these co-factors from their normal locations nuclear localization signal (nls-cre), into the Camk2a-pMM403 vector33 as and to perturb transcription that is mediated by CBP or TAF2C1. previously described34. We injected purified, linearized DNA into pronu- Consistent with this, transcriptional activities of CREB and Elk1, clei of FVB/N oocytes. We chose to use the Camkcre4 line because of its two transcription factors that interact with CBP or TAF2C1, are robust and widespread pattern of Cre expression in the forebrain. To gen- reduced by mutant huntingtin25,26,29. CBP and TAF2C1, how- erate the D1cre line, we isolated a YAC of 140 kb from a C57Bl/6 mouse ever, interact with many transcription factors; thus, it was genomic library35. We introduced the nls-cre ORF into the dopamine unclear which of the transcriptional pathways acting down- receptor-D1A gene (Drd1a) by homologous recombination in yeast. We stream of CBP or TAF2C1 was involved in triggering neurode- assembled a pop-in/pop-out modification vector in the pRS306 (ref. 36) vector using a region of 800 bp showing 5′ homology to Drd1a and a region generation. Our study clearly shows that disruption of the CREB of 580 bp showing 3′ homology to Drd1a. The second region contains a signaling pathway alone is sufficient to produce a neurodegener- unique XbaI site for linearization of the construct. The modified D1cre ative phenotype in the mouse. In light of the reported inhibitory YAC DNA was isolated as described37 and injected into pronuclei of effect of the expanded polyglutamine stretch of huntingtin on FVB/N oocytes. The pattern of Cre expression was identical in four of the CBP function, it is notable that the conserved pattern of degener- five lines obtained. The D1cre line used in this study harbors four copies of ation involves the dorsolateral striatum in both the transgene and was selected because of its robust expression levels. cre Creb1Camkcre4Crem–/– and Creb1D1creCrem–/– models. Striatum is transgenic mice were genotyped by dot-blot analysis of tail DNA. the main region of brain to undergo degeneration in Huntington disease, both in humans and in several rodent models30. It is Immunohistochemistry. We perfused mice with cold 4% paraformalde- therefore probable that interference with CREB-mediated tran- hyde (PFA). Depending on the age of the mice, we either dissected brains scription contributes to neuronal loss in a subset of polygluta- (adult) or post-fixed whole heads (E16.5 and E18.5) overnight in 4% PFA at 4 °C; in either case, we embedded the samples in paraffin wax. We sec- © http://genetics.nature.com Group 2002 Nature Publishing mine diseases such as Huntington disease. tioned the paraffin blocks on a microtome at a thickness of 7 µm, cleared Mice lacking CREB and ATF1 show early developmental arrest the sections of paraffin and rehydrated them through an ethanol dilution 31 and increased cell death at a time when CREM is not yet present . series. We cut postnatal brains at a thickness of 50 µm on a vibratome Increased apoptosis of spermatids, which normally express only (Leica) and further processed floating sections for immunohistochemical CREM, is also responsible for the infertility of Crem–/– male mice14. detection using the VECTASTAIN ABC system (Vector Laboratories) and Together, these observations provide genetic evidence that all three diaminobenzidine (DAB; Sigma) incubation. We used the following pri- CREB family members are crucial for the maintenance of cell via- mary antibodies: polyclonal anti-Creb (N-terminal epitope: H2N-SGAD- bility in a variety of tissues and at various stages of development. NQQSGDAAVTEC-CONH2,1:9,000), polyclonal anti-Cre (Covance Moreover, this study shows that CREB and CREM are crucial for Research Products, 1:3,000), polyclonal anti–Ki-67 (Dianova, 1:100), monoclonal anti-neuN (Chemicon, 1:500), monoclonal anti-GFAP (Sig- the maintenance of neuronal survival in vivo, raising the possibility ma, 1:500), polyclonal anti–cleaved caspase 3 (Cell Signaling, 1:100) and that pharmacological manipulation of the CREB signaling pathway polyclonal anti–mouse Bcl2 (PharMingen, 1:400). may exert positive therapeutic action on both the functional and anatomical deterioration that follows the progression of neurode- Nissl staining. We rehydrated paraffin sections, rinsed them in water, generative disorders. incubated them for 10 min in 0.1% cresyl violet and rinsed them again in water. We destained the slides in 250 ml 96% ethanol containing 5 or 6 Methods drops of acetic acid, then dehydrated and mounted them. Generation of Creb1loxP mice. To generate the nervous system–specific CREB mutant mice, we used homologous recombination in ES cells to TUNEL labeling. We cleared paraffin sections of embryos and rehydrated modify the Creb1 allele such that Creb1 exon 10, encoding the first part of them through an ethanol series. After a water rinse, we incubated the sections the bZIP domain, was flanked by loxP sites. Cre-mediated recombination for 5 min in 20 µg ml–1 proteinase K and for 5 min in 2% H O at room tem- loxP 2 2 of the Creb1 allele leads to a Creb1 null allele that encodes a truncated perature. We carried out staining according to the manufacturer’s instruc- CREB protein devoid of DNA-binding and dimerization domains. This tions using the In Situ Cell Detection Kit (Roche). We detected positive cells truncated protein is unstable; thus, successful recombination results in loss with the VECTASTAIN Elite Kit (Vector Laboratories) and with DAB sub- loxP of CREB. Mice harboring the Creb1 allele were crossed with transgenic strate (Roche), as described for the immunohistochemical staining. mice carrying a transgene for Cre recombinase under the control of the nestin promoter and enhancer16. To generate Creb1NescreCrem–/– mice, we RNase protection assay. We used 5 µg of total RNA for analysis. For the crossed the Creb1loxP mice with Crem–/– mice15. The only two possible probe set, we used the mouse Apo-2 RiboQuant set (Pharmingen), which kinds of breeding pairs that could yield useful numbers of double-mutant was used as the template to generate radiolabeled ([32P]UTP) riboprobes. Creb1NescreCrem–/– (NescreCreb1loxP/loxPCrem–/–) mice were We hybridized the RNA and probes for 16 h at 56 °C, then digested them NescreCreb1+/loxPCrem+/– males crossed with Creb1loxP/loxPCrem–/– females, with RNase A and RNase T1 at 30 °C for 45 min, precipitated them and and Creb1loxP/loxPCrem+/– males crossed with NescreCreb1+/loxPCrem–/– resolved the products on a 6% acrylamide sequencing gel. We carried out females. Male Crem–/– mice are sterile, and both male and female image analysis using a Molecular Dynamics Phosphorimager.

nature genetics • volume 31 • may 2002 53 article

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