白腰文鸟发声行为的神经发育

本文研究了 5～ 15 0日龄雄性白腰文鸟 (Lonchurastriataswinhoei)不同年龄段的声谱变化以及这种变化的神经调制机制。结果如下 :(1)HVC、RA和AreaX三个发声核团的神经联系基本接近成年鸟的水平后 ,幼鸟才开始学习鸣叫 (约 45日龄 ) ;(2 )HVC、RA和AreaX达到成年核团体积时 (约 80日龄 ) ,幼鸟才具有成年雄鸟的鸣叫模式 ;(3)发声控制核团的发育与核团间的神经支配有关 ,而基本不受鸣唱行为的影响 ,HVC、RA和AreaX的最快增长时间段各不相同 ,三个核团随年龄增长而呈现体积增长的显著变化 (one wayANOVA ,P <0 0 5 ) ,但各核团在任意两个时间段的体积差异并不都显著。结果提示 :发声行为产生的时间和发展与发声控制核团的发育、核团间的神经联系有关 ,最终的体积发育程度受内在遗传力的作用 ,同时可能还受神经核团建立正常神经联系时间的影响     

Sex differences in the telencephalic song control circuitry in Bengalese finches (Lonchura striata var. domestica)

Bengalese finches, Lonchura striata, are extremely sexually dimorphic in their singing behavior; males sing complex songs, whereas females do not sing at all. This study describes the developmental differentiation of the brain song system in Bengalese finches. Nissl staining was used to measure the volumes of four telencephalic song nuclei: Area X, HVC, the robust nucleus of the arcopallium (RA), and the lateral portion of the magnocellular nucleus of the anterior nidopallium (LMAN). In juveniles (circa 35 days old), Area X and the HVC were well developed in males, while they were absent or not discernable in females. The RA was much larger in males but barely discernable in females. In males, the volumes of Area X and the RA increased further into adulthood, but that of the HVC remained unchanged. The LMAN volume was greater in juveniles than in adults, and there was no difference in the LMAN volume between the sexes. The overall tendency was similar to that described in zebra finches, except for the volume of the RA, where the degree of sexual dimorphism is larger and the timing of differentiation occurs earlier in Bengalese finches. Motor learning of the song continues until day 90 in zebra finches, but up to day 120 in Bengalese finches. Earlier neural differentiation and a longer learning period in Bengalese finches compared with zebra finches may be related to the more elaborate song structures of Bengalese finches.     

Song-selective auditory input to a forebrain vocal control nucleus in the zebra finch

Neurons in nuclei on the motor pathway for vocalizations in songbirds are known to responses in one such nucleus, robustus archistriatalis (RA), were characterized by making multi-unit recordings in awake and anesthetized adult male zebra finches and in birds that had received lesions of the input to RA from the lateral part of the magnocellular nucleus of the anterior neostriatum (LMAN) or the Higher Vocal Center (HVC). In awake birds, RA neurons have a high level of spontaneous activity and vigorous auditory responses to song stimuli. Significantly greater responses are seen to the bird's own song (BOS) than to BOS played in reverse (REV) or to the songs of conspecifics (CON). Under ketamine-xylazine anesthesia, spontaneous activity is reduced, response latency increases and responses to BOS, REV and CON are indistinguishable. Responses obtained under urethane anesthesia are similar to those seen in awake birds. Thus, the pattern and selectivity of auditory responses in RA depend on the animal's state. Auditory responses in RA are qualitatively unchanged following lesion of the input to RA from LMAN, indicating that this pathway is not required for the sensory processing that underlies the preference for BOS on the vocal production pathway. Our results show that an input other than that from LMAN must be primarily responsible for auditory responses in RA. The direct projection form HVC is the most likely pathway by which song selective auditory information arrives in RA, since lesioning HVC abolished auditory responses in RA. © 1993 John Wiley & Sons, Inc.     

Forebrain circuits underlying the social modulation of vocal communication signals

Across vertebrate species, signalers alter the structure of their communication signals based on the social context. For example, male Bengalese finches produce faster and more stereotyped songs when directing song to females (female‐directed [FD] song) than when singing in isolation (undirected [UD] song), and such changes have been found to increase the attractiveness of a male's song. Despite the importance of such social influences, little is known about the mechanisms underlying the social modulation of communication signals. To this end, we analyzed differences in immediate early gene (EGR‐1) expression when Bengalese finches produced FD or UD song. Relative to silent birds, EGR‐1 expression was elevated in birds producing either FD or UD song throughout vocal control circuitry, including the interface nucleus of the nidopallium (NIf), HVC, the robust nucleus of the arcopallium (RA), Area X, and the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Moreover, EGR‐1 expression was higher in HVC, RA, Area X, and LMAN in males producing UD song than in males producing FD song, indicating that social context modulated EGR‐1 expression in these areas. However, EGR‐1 expression was not significantly different between males producing FD or UD song in NIf, the primary vocal motor input into HVC, suggesting that context‐dependent changes could arise de novo in HVC. The pattern of context‐dependent differences in EGR‐1 expression in the Bengalese finch was highly similar to that in the zebra finch and suggests that social context affects song structure by modulating activity throughout vocal control nuclei. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 47–63, 2016     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

白腰文鸟发声行为的性别差异及其机制

通过声谱分析,研究了５－１２０日龄雌、雄白腰文鸟（Lonchura striata swinhoei)的声谱变化,及该时段３个主要发声控制核团）ＨＶＣ、ＲＡ、Ａrea X)体积、睾丸（睾酮）的相应改变。结果如下：①４５日龄以前,雌雄鸟只能发出简单鸣叫（call),鸣声基本不会鸣唱。②雄性ＨＶＣ,ＲＡ,ＡreaX体积均比雌性大２－６部。３个核团的大小发育不完全一致。各核团的快速生长期与鸣唱学习的主要时段（６０－１２０日龄）不同步,说明核团的个体发育可能不完全受发声行为的影响。③睾丸的充分发育（１２０日龄后）及血液中具有较高的睾酮水平是雄鸟发出成熟鸣唱语句的重要条件。     

Minimal BH3 peptides promote cell death by antagonizing anti-apoptotic proteins

The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.     

Female marsh wrens do not provide evidence of anatomical specializations of song nuclei for perception of male song

In songbirds the forebrain nuclei HVC (high vocal center) and RA (robust nucleus of the archistriatum) are larger in individuals or species that produce larger song repertoires, but the extent to which the size of these nuclei reflects a need for either producing or perceiving large repertoires is unknown. We, therefore, tested the hypothesis that species differences in the size of song nuclei reflect a commitment of “brain space” to the perceptual processing of conspecific song. The two species of marsh wren (Cistothorus palustris western and eastern) provide a good test case. Western males produce larger song repertoires, and have larger HVC and RA than do eastern males. Female marsh wrens do not sing, and if they use their song nuclei to assess conspecific male song repertoires, then we predicted that measurable cellular and nuclear parameters of HVC and RA would be greater in western than eastern female wrens. For males we confirmed that the volumes of HVC and RA, and cellular parameters of HVC, are greater in western than in eastern birds. These nuclei were also considerably larger in males than in conspecific females. Western and eastern female wrens, however, did not differ in any measured parameters of HVC or RA. Females of these wren species thus do not provide any direct evidence of anatomical specializations of song nuclei for the perceptual processing of conspecific male song. 1994 John Wiley & Sons, Inc.     

新纹状体巨细胞核外侧部（LMAN）与鸣禽鸣唱学习可塑性

鸣禽鸣唱控制系统的前端脑通路（anterior forebrain pathway, AFP）在鸣唱学习中发挥着重要作用。新纹状体巨细胞核外侧部（lateral magnocellular nucleus of the anterior neostriatum, LMAN）是AFP的最后一级输出核团,AFP中的信号通过LMAN传导到弓状皮质栎核（robust nucleus of the arcopallium, RA）,与高级发声中枢（high vocal centre,HVC）共同调节RA的活动,从而影响鸣禽的发声行为。LMAN可能通过其与RA的单突触连接来影响鸣唱可塑性。文章对近年来LMAN在鸣唱学习可塑性方面的研究进行综述。     

Characterization of the G-protein-coupled membrane-bound estrogen receptor GPR30 in the zebra finch brain reveals a sex difference in gene and protein expression

Estradiol‐induced structural dimorphisms exist in the songbird brain. However, how they arise is not clear since there is a scarce distribution of ERα and lack of ERβ in song control nuclei. This suggests that other receptors are involved. The G‐protein coupled membrane‐bound estrogen receptor, GPR30, is a candidate but has never been investigated in songbirds. In this study, we characterized its gene and protein in the zebra finch brain. Analysis of the putative GPR30 protein sequence revealed a strong similarity to avian and mammalian homologues. Quantitative PCR indicated that the gene was elevated in the telencephalon of both sexes from posthatching day (P) 15 to P45, with a male‐biased sex difference at P21 and P30. In comparison, expression at younger posthatching ages and in adults was significantly less. At P21, GRP30 protein was widespread, nonuniform, and overlapped with song control nuclei. Of particular interest, the number of immunoreactive cells was greatest in HVC and RA, but less in LMAN and Area X. Labeling in HVC was also dimorphic; with more cells present in males than in females. In parallel with the gene, by adulthood, protein expression was reduced across most brain regions. Taken together these data suggest that GPR30 may contribute to differences in song system development by mediating dimorphic responses to estrogens. In addition, the extensive protein distribution indicates that it may also have a role in general brain development in both sexes. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Seasonal changes in patterns of gene expression in avian song control brain regions

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.     

鱼类和哺乳类Bcl-2家族蛋白的研究进展

细胞凋亡, 即细胞程序性死亡, 在多细胞生物的发育和稳态调控过程中发挥关键作用。Bcl-2家族蛋白是凋亡过程中的主要调控因子, 关于Bcl-2家族蛋白在凋亡过程中的功能及其作用机制一直是研究的热点。已有研究显示Bcl-2家族蛋白不仅作用于线粒体引发凋亡, 并且参与了包括对细胞内质网Ca2+的调控、DNA损伤的修复及与自噬的相互作用等多种反应, 从多方面对细胞的生存状态进行调控。Bcl-2家族蛋白保守存在于脊椎动物和无脊椎动物中, 其功能在进化中存在异同。文章以高等脊椎动物(哺乳动物)和低等脊椎动物(硬骨鱼类)为代表, 总结了近年来Bcl-2家族蛋白在调控宿主凋亡与自噬、DNA损伤及新陈代谢等方面取得的最新进展。该研究为深入了解鱼类和哺乳类Bcl-2家族蛋白的功能和作用机制提供了重要参考。     

Neurotensin and neurotensin receptor 1 mRNA expression in song‐control regions changes during development in male zebra finches

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine‐related measures change throughout development in song‐control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song‐control regions of male zebra finches in four stages of the song learning process: pre‐subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre‐subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018     

Early condition, song learning, and the volume of song brain nuclei in the zebra finch (Taeniopygia guttata)

Songbirds are an important model system for the study of the neurological bases of song learning, but variation in song learning accuracy and adult song complexity remains poorly understood. Current models of sexual selection predict that signals such as song must be costly to develop or maintain to constitute honest indicators of male quality. It has been proposed that reductions of nestling condition during song development might limit the expression of song learning. Adult song could thus act as an indicator of early stress as only males that enjoy good condition during development could learn accurately and sing long songs or large repertoires. We tested this hypothesis in the zebra finch by modifying early condition through cross-fostering chicks to small, medium, and large broods. Song learning was very accurate and was found to reflect very closely tutor song characteristics and to depend on the number of males in the tutoring group. Although the brood size manipulation strongly affected several measures of nestling condition and adult biometry, we found no relationship between early condition and song learning scores or song characteristics. Similarly, brain mass and high vocal center (HVC), robust nucleus of the arcopallium (RA), and lateral magnocellular nucleus of the anterior nidopallium (LMAN) volumes did not covary with nestling condition and growth measurements. We found no significant relationship between song repertoire size and HVC and RA volumes, although there was a nonsignificant trend for HVC to increase with increasing proportion of learnt elements in a song. In conclusion, the results provide no evidence for song learning to be limited by nestling condition during the period of nutritional dependence from the parents in this species.     

HVC microlesions do not destabilize the vocal patterns of adult male zebra finches with prior ablation of LMAN

The songs of adult male zebra finches (Taeniopygia guttata) arise by an integration of activity from two neural pathways that emanate from the telencephalic nucleus HVC (proper name). One pathway descends directly from HVC to the vocal premotor nucleus RA (the robust nucleus of the arcopallium) whereas a second pathway descends from HVC into a basal ganglia circuit (the anterior forebrain pathway, AFP) that also terminates in RA. Although HVC neurons that project directly to RA outnumber those that contribute to the AFP, both populations are distributed throughout HVC. Thus, partial ablation (microlesion) of HVC should damage both pathways in a proportional manner. We report here that bilateral HVC microlesions in adult male zebra finches produce an immediate loss of song stereotypy from which birds recover, in some cases within 3 days. The contribution of the AFP to the onset of song destabilization was tested by ablating the output nucleus of this circuit (LMAN, the lateral magnocellular nucleus of the anterior nidopallium) prior to bilateral HVC microlesions. Song stereotypy was largely unaffected. Together, our findings suggest that adult vocal production involves nonproportional integration of two streams of neural activity with opposing effects on song--HVC's direct projection to RA underlies production of stereotyped song whereas the AFP seems to facilitate vocal variation. However, the rapid recovery of song in birds with HVC microlesions alone suggests the presence of dynamic corrective mechanisms that favor vocal stereotypy.     

Variation in the volume of zebra finch song control nuclei is heritable: developmental and evolutionary implications.

In many songbird species, females prefer males that sing a larger repertoire of syllables. Males with more elaborate songs have a larger high vocal centre (HVC) nucleus, the highest structure in the song production pathway. HVC size is thus a potential target of sexual selection. Here we provide evidence that the size of the HVC and other song production nuclei are heritable across individual males within a species. In contrast, we find that heritabilities of other nuclei in a song-learning pathway are lower, suggesting that variation in the sizes of these structures is more closely tied to developmental and environmental differences between individuals. We find that evolvability, a statistical measure that predicts response to selection, is higher for the HVC and its target for song production, the robustus archistriatalis (RA), than for all other brain volumes measured. This suggests that selection based on the functions of these two structures would result in rapid major shifts in their anatomy. We also show that the size of each song control nucleus is significantly correlated with the song related nuclei to which it is monosynaptically connected. Finally, we find that the volume of the telencephalon is larger in males than in females. These findings begin to join theoretical analyses of the role of female choice in the evolution of bird song to neurobiological mechanisms by which the evolutionary changes in behaviour are expressed.     

Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Anatomically Discrete Sex Differences in Neuroplasticity in Zebra Finches as Reflected by Perineuronal Nets

Large morphological sex differences in the vertebrate brain were initially identified in song control nuclei of oscines. Besides gross differences between volumes of nuclei in males and females, sex differences also concern the size and dendritic arborization of neurons and various neurochemical markers, such as the calcium-binding protein parvalbumin (PV). Perineuronal nets (PNN) of the extracellular matrix are aggregates of different compounds, mainly chondroitin sulfate proteoglycans, that surround subsets of neurons, often expressing PV. PNN develop in zebra finches song control nuclei around the end of the sensitive period for song learning and tutor deprivation, known to delay the end of the song learning sensitive period, decreases the numbers of PNN in HVC. We demonstrate here the existence in zebra finches of a major sex difference (males > females) affecting the number of PNN (especially those surrounding PV-positive cells) in HVC and to a smaller extent the robust nucleus of the arcopallium, RA, the two main nuclei controlling song production. These differences were not present in Area X and LMAN, the lateral magnocellular nucleus of the anterior nidopallium. A dense expression of material immunoreactive for chondroitin sulfate was also detected in several nuclei of the auditory and visual pathways. This material was often organized in perineuronal rings but quantification of these PNN did not reveal any sex difference with the exception that the percentage of PNN surrounding PV-ir cells in the dorsal lateral mesencephalic nucleus, MLd, was larger in females than in males, a sex difference in the opposite direction compared to what is seen in HVC and RA. These data confirm and extend previous studies demonstrating the sex difference affecting PNN in HVC-RA by showing that this sex difference is anatomically specific and does not concern visual or auditory pathways.     

Distribution and onset of retinaldehyde dehydrogenase (zRalDH) expression in zebra finch brain: lack of sex difference in HVC and RA at early posthatch ages

Using in situ hybridization to detect the expression of the retinoic acid synthesizing enzyme (retinaldehyde dehydrogenase: zRalDH) mRNA, we mapped the distribution of its expression in adult zebra finch brain. In the neural song circuit, strong expression was found in high vocal center (HVC), para-HVC, and at a very low level in the robust nucleus of the arcopallium (RA). The expression in HVC and RA was found in both males and females. Outside of the song system, major areas of expression were in medial nidopallium (N), hyperpallium apicale (HA), mesopallium ventrale (MV), taenial amygdala (TnA), cerebellar Purkinje cells, and nucleus isthmo-opticus (IO). In nestlings, we found zRalDH mRNA expression in HVC and RA as early as posthatch day 4 or 5 (P4-5), although the expression varied among individuals. Thus, retinoic acid synthesis in HVC and RA could participate in song system formation and development. However, we found no sex difference in volume or intensity of zRalDH and androgen receptor (AR) expression in HVC and RA at P11 prior to the development of significant size dimorphisms in these nuclei. The size of HVC in females at P11 defined by zRalDH expression was greater than that in adult females, suggesting that HVC might experience net cell loss between P11 and adulthood.     

The Bcl-2 Homology Domain 3 (BH3)-only Proteins Bim and Bid Are Functionally Active and Restrained by Anti-apoptotic Bcl-2 Family Proteins in Healthy Liver

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.