BOLD’s role in barcode data management and analysis: a response

DNA barcoding is a very effective tool for the identification of specimens when a carefully validated and taxonomically comprehensive library of reference DNA barcodes is available. Libraries meeting this criterion are now available for some taxonomic groups in some geographic regions, provoking their use as a tool for the identification of samples that would otherwise remain as unknowns. In this article, we emphasize the need for caution in the interpretation of identifications based on a reference library with entries that have seen limited validation. We also emphasize the need for the deposition of sequence records for ‘unknowns’ so that presumptive identifications can be tested by other researchers and updated as the barcode reference library gains increased coverage and validation.     

RNA在生物学的重要地位及其作为常规治疗药物的研究进展

20多年来的研究发现,RNA除了具有如tRNA、rRNA参与蛋白质生物合成的基本功能外,细胞内还存在许多种类的RNA,它们执行着不同的功能,在细胞内生物化学反应及机体发育调控过程中发挥着重要作用。正因为RNA功能多样性,在体内、体外开展的众多实验表明,RNA或其修饰形式可以抑制基因的表达。该文将探讨RNA在常规基因治疗中的研究。     

The cell biology of autophagy in metazoans: a developing story

The cell biological phenomenon of autophagy (or ;self-eating') has attracted increasing attention in recent years. In this review, we first address the cell biological functions of autophagy, and then discuss recent insights into the role of autophagy in animal development, particularly in C. elegans, Drosophila and mouse. Work in these and other model systems has also provided evidence for the involvement of autophagy in disease processes, such as neurodegeneration, tumorigenesis, pathogenic infection and aging. Insights gained from investigating the functions of autophagy in normal development should increase our understanding of its roles in human disease and its potential as a target for therapeutic intervention.     

The quest for adaptive evolution: a theoretical challenge in a maze of data

Advances in sequencing technology have brought opportunities to refine our searches for adaptive evolution and to address and identify new questions regarding how adaptive evolution has shaped genomic diversity. Recent theoretical developments incorporate demographic and complex selective histories into tests of non-neutral evolution, thereby significantly improving our power to detect selection. These analyses combined with large data sets promise to identify targets of selection for which there was no a priori expectation. Moreover, they contribute to elucidate the role selection has played in shaping diversity in transposable elements, conserved noncoding DNA, gene family size, and other multicopy features of genomes.     

Character identification in evolutionary biology: The role of the organism

Summary In this article we argue that an organismic perspective in character identification can alleviate a structural deficiency of mathematical models in biology relative to the ones in the physical sciences. The problem with many biological theories is that they do not contain the conditions of their validity or a method of identifying objects that are appropriate instances of the models. Here functionally important biological characters are introduced as conceptual abstractions derived within the context of an ontologically prior object, such as a cell or an organism. To illustrate this approach, we present an analytical method of character decomposition based on the notion of the quasi-independence of traits. Two cases are analyzed: context dependent units of inheritance and a model of character identification in adaptive evolution. We demonstrate that in each case the biological process as represented by a mathematical theory entails the conditions for the individualization of characters. Our approach also requires a conceptual re-orientation in the way we build biological models. Rather than defining a set of biological characters a priori, functionally relevant characters are identified in the context of a higher level biological process.     

On the limitations of standard statistical modeling in biological systems: A full Bayesian approach for biology

One of the most important scientific challenges today is the quantitative and predictive understanding of biological function. Classical mathematical and computational approaches have been enormously successful in modeling inert matter, but they may be inadequate to address inherent features of biological systems. We address the conceptual and methodological obstacles that lie in the inverse problem in biological systems modeling. We introduce a full Bayesian approach (FBA), a theoretical framework to study biological function, in which probability distributions are conditional on biophysical information that physically resides in the biological system that is studied by the scientist.     

The future of metabolic engineering and synthetic biology: towards a systematic practice

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.     

Physics and the canalization of morphogenesis: a grand challenge in organismal biology

Morphogenesis takes place against a background of organism-to-organism and environmental variation. Therefore, fundamental questions in the study of morphogenesis include: How are the mechanical processes of tissue movement and deformation affected by that variability, and in turn, how do the mechanic of the system modulate phenotypic variation? We highlight a few key factors, including environmental temperature, embryo size and environmental chemistry that might perturb the mechanics of morphogenesis in natural populations. Then we discuss several ways in which mechanics-including feedback from mechanical cues-might influence intra-specific variation in morphogenesis. To understand morphogenesis it will be necessary to consider whole-organism, environment and evolutionary scales because these larger scales present the challenges that developmental mechanisms have evolved to cope with. Studying the variation organisms express and the variation organisms experience will aid in deciphering the causes of birth defects.     

The biological standard of living in Taiwan under Japanese occupation

This paper presents evidence on the biological standard of living in Taiwan from 1842 to 1931 using Taiwanese height and weight data collected by the Japanese authorities from 1921 to 1931. This study shows that in the late Ch'ing adult heights were not increasing over time, while the adult heights of those born after the Japanese takeover did begin to increase rapidly. Evidence from children's heights confirms that this growth in height continued through the 1920s. The body mass index of Taiwanese, however, did not increase in the 1920s. By most measures, the biological standard of living was better in the north of the island. Comparison with modern data shows that heights have continued to increase.     

Boning up on autophagy: The role of autophagy in skeletal biology

From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton.     

The role of habitat quality in fragmented landscapes: a conceptual overview and prospectus for future research

There is increasing empirical evidence that the quality of habitat patches (determined by either habitat degradation or natural heterogeneity in the quality of habitat) plays an important role in determining species distribution patterns and in regulating spatial dynamics in fragmented landscapes. However, to date, most of the debate has focused on whether or not to include habitat variables in fragmentation studies, and we still lack general conclusions as well as standard and robust research approaches. In this paper we show how a weak conceptualization of “patch quality” and the inappropriate choice of target surrogate variables (e.g., density is often used as an indicator of patch quality) have mainly produced case-specific results, rather than general conclusions. We then identify weaknesses in the inclusion of habitat quality measurements within fragmentation studies. In particular, we focus on: (1) the lack of appropriate experimental design, outlining how few studies have actually included a gradient of habitat quality in their sample; (2) the lack of fundamental information provided (e.g., lack of standard outputs), which in turn hampers the possibility of carrying out meta-analyses. We finally synthesize available knowledge from empirical studies and highlight the different conceptual frameworks needed for patch occupancy versus patch use studies.     

Similarity measures for structured data: a general framework and some applications to vegetation data

Although there are many measures of similarity existing in the phytosociological literature, these almost all apply to data for which the describing attributes have only single values. In many cases, however, there can be a richer structure in the attribute values, either directly from the nature of the attributes or derived from relationships between the stands. In this paper, I first examine a range of possible sources of such structure in phytosociological data, and then propose a similarity measure sufficiently general to be applicable to all the variant types. Finally I present some examples of applying such measures to frequency data from tropical grasslands and to successional data from subtropical rain forest.     

Species separation and identification in the Enchytraeidae (Oligochaeta, Annelida): combining morphology with general protein data

In the Enchytraeidae, species separation and identification is often problematic due to high morphological similarity of closely related species and considerable intraspecific variability of crucial characteristics. Immature specimens are almost undeterminable. To meet these difficulties, this paper recommends the consulting of general protein patterns as exhibited by non-specific silver-staining after isoelectric focusing. A method is presented which allows the successful inclusion of protein data in taxonomic studies and field surveys. Two examples from an investigation on Fridericia field populations show that, with the help of general protein pattern analysis, a clear taxonomic decision on the identity of morphologically aberrant forms and of juvenile specimens as well can be achieved with comparatively little expenditure of time. A combined use of morphological and protein data for taxonomic purposes is suggested.     

The biological standard of living in the two Koreas

Height data of North Korean escapees are analyzed to assess changes in their biological standard of living. In contrast to the population of South Korea, as well as to that of most of the rest of the world, North Koreans did not experience an increase in physical stature during the second half of the 20th century. The divergence between the height of North- and South-Koreans began among the birth cohorts of the late 1940s and became increasingly pronounced thereafter. This is an indication of the adverse socio-economic circumstances prevailing in the northern part of the Korean peninsula.     

Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology

Molecular morphology seeks to integrate the traditional morphologic criteria of surgical pathology with immunohistochemical and in situ hybridization techniques that allow demonstration of a variety of molecules, proteins, RNA and DNA in a tissue section. While immunohistochemistry has proven to be successful for demonstrating lineage related biomarkers of value for diagnosis and classification of tumors, concerns have been raised periodically about validation of reagents, overall reproducibility of the staining method, and interpretation of results. These concerns have been heightened by the burgeoning interest in prognostic markers, where the question extends beyond a relatively simple positive or negative result to an absolute need for quantification of the staining result; not only is it positive, but how much is there? In this presentation at the Annual Meeting of the Biological Stain Commission in June, 2005, I advocate a total test approach that requires systematic attention to pre-analytic, analytic, and post-analytic issues. The approach encompasses all aspects of test performance from specimen acquisition, through fixation, antigen retrieval, processing, staining, interpretation, and reporting of results. A similar systematic approach also may be adopted for in situ hybridization methods, which have performance requirements that in many ways parallel immunohistochemistry.     

New tools for the investigations of Neuro-AIDS at a molecular level: The potential role of data-mining

Cognitive impairment represents the most significant and devastating neurological complication associated with HIV infection. Despite recent advances in our knowledge of the clinical features, pathogenesis, and molecular aspects of HIV-related dementia, current diagnostic strategies are associated with significant limitations. It has been suggested that the use of some biomarkers may assist researchers and clinicians in predicting the onset of the disease process and in evaluating the effects of new therapies. However, the large number of chemicals and metabolic pathways involved in the pathogenesis of neurodegeneration, warrants the development of novel approaches to integrate this huge amount of data. The contribution of theoretical disciplines, such as bioinformatics and data-mining, may be useful for testing new hypotheses in diagnosis and patient-centered treatment interventions.     

Cell biology in neuroscience: Death of developing neurons: New insights and implications for connectivity

The concept that target tissues determine the survival of neurons has inspired much of the thinking on neuronal development in vertebrates, not least because it is supported by decades of research on nerve growth factor (NGF) in the peripheral nervous system (PNS). Recent discoveries now help to understand why only some developing neurons selectively depend on NGF. They also indicate that the survival of most neurons in the central nervous system (CNS) is not simply regulated by single growth factors like in the PNS. Additionally, components of the cell death machinery have begun to be recognized as regulators of selective axonal degeneration and synaptic function, thus playing a critical role in wiring up the nervous system.

Why do so many neurons die during development?

Programmed cell death occurs throughout life, as cell turnover is part of homeostasis and maintenance in most organs and tissues. The situation in the nervous system is principally different, as the vast majority of neurons undergo their last round of cell division early in development. Soon after exiting the cell cycle, neurons start elongating axons to innervate their targets. It is during this period that they are highly susceptible to undergo programmed cell death: a large percentage, as much as 50% in several ganglia in the peripheral nervous system (PNS) as well as in various central nervous system (CNS) areas, is eliminated around the time that connections are being made with other cells. Later in development, the propensity of neurons to initiate apoptosis progressively decreases. The likelihood for a neuron to undergo apoptosis seems to be determined by a tightly regulated apoptotic machinery (summarized in Fig. 1). Therefore, modulation of the expression levels or the activity of components of this apoptotic balance changes the sensitivity to death-promoting cues, allowing temporal restriction of cell death.Open in a separate windowFigure 1.Core components of the apoptotic machinery. The likelihood that a neuron undergoes apoptosis is determined by the interplay of the tightly interlinked apoptotic machinery, many components of which are highly conserved between species. The critical, and often terminal, step in programmed cell death is the proteolytic activation of the executor caspases (such as caspase 3, 6, 7) by the initiator caspases (i.e., caspase 8, 9, and 10; Riedl and Salvesen, 2007). In mammalian cells, initiation of the executor caspases is regulated by two distinct protein cascades: the intrinsic pathway, also known as the mitochondrial pathway, and the extrinsic pathway. The intrinsic pathway integrates a number of intra- and extracellular signal modalities, such as redox state (for example, the reactive oxygen species; Franklin, 2011), DNA damage (Sperka et al., 2012), ER stress (Puthalakath et al., 2007) and growth factor deprivation (Deckwerth et al., 1998; Putcha et al., 2003; Bredesen et al., 2005), or activation of the p75NTR neurotrophin receptor by pro-neurotrophins (Nykjaer et al., 2005). The stressors converge onto pro- and anti-apoptotic members of the Bcl-2 protein family (for example: BCL-2, BCL-X_l, BAX, and tBID; Youle and Strasser, 2008). These proteins regulate the release of cytochrome c from mitochondria, which activates the initiator caspase 9 through Apaf1 (Riedl and Salvesen, 2007). The extrinsic pathway links activation of ligand-bound death receptors (such as Fas/CD95 and TNFR) to the initiator caspase 8 and 10, through formation of the death-inducing signaling complex (DISC; LeBlanc and Ashkenazi, 2003; Peter and Krammer, 2003). Together with additional regulatory elements (including the Inhibitors of apoptosis proteins [IAP]; Vaux and Silke, 2005) and cFLIP (Scaffidi et al., 1999; Wang et al., 2005), the apoptotic machinery forms a balance that determines the propensity of the neuron to undergo apoptosis.Programmed cell death eliminates many neurons during development, even in organisms comprised of only few cells, such as Caenorhabditis elegans. As neurons and their targets are initially separated, it is possible that the initial generation of an overabundance of neurons is simply part of a mechanism to ensure that distal targets are adequately innervated (Oppenheim, 1991; Conradt, 2009; Chen et al., 2013). In various tissues other than the nervous system, programmed cell death is used to eliminate cells that are no longer needed, defective, or harmful to the function of the organism. However, there is strong evidence that the elimination of superfluous neurons in the developing nervous system is not essential. For example, early work in C. elegans revealed that preventing programmed cell death does not result in significant behavioral alterations (Ellis and Horvitz, 1986). In the C57BL/6 mouse strain, deletion of the executor caspases 3 and 7 (Fig. 1) has a remarkably limited neuropathological and morphological impact in the CNS (Leonard et al., 2002; Lakhani et al., 2006) compared with the 129X1/SvJ strain, in which deletion of these caspases causes neurodevelopmental defects (Leonard et al., 2002). Similar conclusions were reached by blocking the Bcl-2–associated X protein (BAX)–dependent pathway in many neuronal populations, including motoneurons (Buss et al., 2006a). A recent study in the developing retina showed that in mice lacking the central apoptotic regulator BAX, the normal mosaic distribution of intrinsically photosensitive retinal ganglion cells (ipRGCs) was perturbed (Chen et al., 2013). Although this abnormal distribution is dispensable for the intrinsic photosensitivity of the ipRGCs, it is required for establishing proper connections to other neurons in the retina, which is necessary for rod/cone photo-entrainment (Chen et al., 2013). Even though this finding highlights a physiological role for programmed cell death in the CNS, the functional consequences remain rather underwhelming in the face of a process that eliminates such large numbers of neurons (Purves and Lichtman, 1984; Oppenheim, 1991; Miller, 1995; Gohlke et al., 2004). It thus appears that apoptotic removal of the surplus neurons generated during development mainly serves the purpose to optimize the size of the nervous system to be minimal, but sufficient.

A molecular substrate for the neurotrophic theory

Quantitatively, programmed cell death of neurons in the PNS and CNS is most dramatic when neurons start contacting the cells they innervate. Because experimental manipulations such as target excision typically lead to the death of essentially all innervating neurons (Oppenheim, 1991), the concept emerged that the fate of developing neurons is regulated by their targets. This notion is often referred to as the “neurotrophic theory” (Hamburger et al., 1981; Purves and Lichtman, 1984; Oppenheim, 1991), but it is important to realize that it evolved in the absence of direct mechanistic or molecular support (Purves, 1988). Originally described as a diffusible agent promoting nerve growth, the eponymous NGF later provided a strong and very appealing molecular foundation for this theory (Korsching and Thoenen, 1983; Edwards et al., 1989; Hamburger, 1992). The tyrosine kinase receptor tropomyosin receptor kinase A (TrkA), which was initially identified as an oncogene (Martin-Zanca et al., 1986), was fortuitously discovered to be the critical receptor necessary for the prevention of neuronal cell death by NGF (Klein et al., 1991). Both the remarkable expression pattern of TrkA in NGF-dependent neurons and the onset of its expression during development (Martin-Zanca et al., 1990) provided further additional support for the neurotrophic theory. However, for a surprisingly long time, the question was not asked as to why only specific populations of neurons strictly depend on NGF for survival, while others do not. Indeed, it was only recently shown that TrkA causes cell death of neurons by virtue of its mere expression, and that this death-inducing activity is prevented by addition of NGF (Nikoletopoulou et al., 2010). These findings thus indicate that TrkA acts as a “dependence receptor,” a concept introduced after observations that various cell types die when receptors are expressed in the absence of their cognate ligands (Bredesen et al., 2005; Tauszig-Delamasure et al., 2007). Accordingly, embryonic mouse sympathetic or sensory neurons survive in the absence of NGF when TrkA is deleted (Nikoletopoulou et al., 2010). The closely related neurotrophin receptor TrkC also acts as a dependence receptor (Tauszig-Delamasure et al., 2007; Nikoletopoulou et al., 2010). Here, it is interesting to note a series of older, convergent results indicating that deletion of neurotrophin-3 (NT3), the TrkC ligand, leads to a significantly larger loss of sensory and sympathetic neurons in the PNS than the deletion of TrkC (Tessarollo et al., 1997). This phenotypic discrepancy fits well with the idea that inactivation of the ligand of a dependence receptor is expected to yield a more profound phenotype than inactivation of the receptor itself (Tauszig-Delamasure et al., 2007). How TrkA and TrkC induce apoptosis remains to be fully elucidated. It seems that proteolysis is involved, either of TrkC itself (Tauszig-Delamasure et al., 2007), as was suggested for other dependence receptors (Bredesen et al., 2005), or by the proteolysis of the neurotrophin receptor p75NTR, which associates with both TrkA and TrkC (Fig. 2; Nikoletopoulou et al., 2010). Surprisingly, although TrkA and TrkC cause cell death, the structurally related TrkB receptor does not (Nikoletopoulou et al., 2010), a difference that appears to be accounted for by their differential localization in the cell membrane. TrkA and TrkC colocalize with p75NTR in lipid rafts, whereas TrkB, which also associates with p75NTR (Bibel et al., 1999), is excluded from lipid rafts (Fig. 2; unpublished data). Interestingly, the transmembrane domains of TrkA and TrkC are closely related, and differ clearly from that of TrkB. It turns out that a chimeric protein of TrkB with the transmembrane domain of TrkA causes cell death, which can be prevented by the addition of the TrkB ligand brain-derived neurotrophic factor (BDNF; unpublished data). The suggestion that the lipid raft localization of TrkA and TrkC is important for their death-inducing function is in line with a number of reports indicating that certain apoptotic proteins preferentially localize in lipid rafts in the plasma membrane. After activation of the extrinsic apoptosis pathway, translocation of the activated receptors to lipid rafts in the membrane is required for assembling the death-inducing signaling complex (DISC; Davis et al., 2007; Song et al., 2007). Indeed, regulators of the extrinsic pathway (e.g., cFLIP; Fig. 1) prevent this translocation, explaining how they attenuate cell death induction (Song et al., 2007). Similarly, the localization of the dependence receptor DCC (deleted in colorectal cancer) in lipid rafts is a prerequisite for its pro-apoptotic activity in absence of its ligand, Netrin-1 (Furne et al., 2006).Open in a separate windowFigure 2.TrkA and TrkC as dependence receptors: mode of action and contrast with TrkB. All Trk receptors associate with the pan-neurotrophin receptor p75NTR (Bibel et al., 1999). A critical step in the induction of apoptosis by TrkA is the release of the intracellular death domain of p75NTR by the protease γ-secretase (Nikoletopoulou et al., 2010), which is localized in lipid rafts (Urano et al., 2005). Our membrane fractionation studies indicate that while TrkA and TrkC associate with p75NTR in lipid rafts, TrkB associated with p75NTR is excluded from this membrane domain (unpublished data). The 24–amino acid transmembrane domain of the Trk receptors may be responsible for this differential localization (see text).Despite the fact that TrkB does not act as a dependence receptor, its activation by BDNF is required for the survival of several populations of cranial sensory neurons (Ernfors et al., 1995; Liu et al., 1995). It appears that other death-inducing receptors predispose these neurons to be eliminated, such as p75NTR, which is expressed at high levels in some of these ganglia, or TrkC in vestibular neurons (Stenqvist et al., 2005). This latter case is of special interest, as NT3 is known not to be required for the survival of these neurons (Stenqvist et al., 2005). In addition to inducing apoptosis in the absence of their ligand, TrkA and TrkC have long been recognized to have a pro-survival function similar to TrkB, as can be inferred from the loss of specific populations of peripheral sensory neurons in mutants lacking these receptors (Klein et al., 1994; Smeyne et al., 1994).

Cell death in the CNS

Although TrkA is primarily expressed in peripheral sympathetic and sensory neurons, it is also found in a small population of cholinergic neurons in the basal forebrain (Sobreviela et al., 1994), a proportion of which requires NGF for survival (Hartikka and Hefti, 1988; Crowley et al., 1994; Müller et al., 2012). Selective deletion of TrkA was recently shown not to cause the death of these neurons (Sanchez-Ortiz et al., 2012). This supports the notion that TrkA acts as a dependence receptor for this small population of CNS neurons, like for peripheral sensory and sympathetic neurons. TrkA activation by NGF is essential for the maturation, projections, and function of these neurons (Sanchez-Ortiz et al., 2012), as was previously described for sensory neurons in the PNS as well (Patel et al., 2000).Whether or not receptors other than TrkA act as dependence receptors in the CNS is an important open question, particularly because TrkB, which is expressed highly by most CNS neurons, does not act as a dependence receptor (Nikoletopoulou et al., 2010). In retrospect, the structural similarities between TrkA and TrkB, just like those between NGF and BDNF (Barde, 1989), have substantially misled the field by suggesting that BDNF would act in the CNS like NGF in the PNS. Adding to the confusion were early findings showing that BDNF supports the growth of spinal cord motoneurons in vitro or in vivo after axotomy (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). However, in the absence of lesion, deletion of BDNF does not lead to significant losses of neurons in the developing or adult CNS (Ernfors et al., 1994a; Jones et al., 1994; Rauskolb et al., 2010), unlike the case in some populations of PNS neurons. The poor correlation of the role of BDNF in CNS development and in axotomy and in vitro experiments is surprising, especially because the role of NGF in vivo could in essence be recapitulated by in vitro experiments. Although the reasons for this discrepancy are not fully understood, the strong up-regulation of death-inducing molecules such as p75NTR after axotomy (Ernfors et al., 1989) may be a part of the explanation. At present, most of the growth factors promoting the survival of PNS neurons fail to show significant survival properties for developing neurons in the CNS, as for example was shown for NT3 (Ernfors et al., 1994b; Fariñas et al., 1994), glial cell line–derived neurotrophic factor (GDNF; Henderson et al., 1994), ciliary neurotrophic factor (CNTF; DeChiara et al., 1995), and several others.In the developing CNS, neuronal activity and neurotransmitter input seem to play a more significant role than single growth factors in regulating neuronal survival. In particular, it has been known for a long time that blocking synaptic transmission at the neuromuscular junction has a pro-survival effect on spinal cord motoneurons (Pittman and Oppenheim, 1978; Oppenheim et al., 2008). By contrast, surgical denervation of afferent connections leads to increased apoptosis of postsynaptic neurons (Okado and Oppenheim, 1984), whereas inhibiting glycinergic and GABAergic synaptic transmission has both pro- and anti-apoptotic effects on motoneurons (Banks et al., 2005). Throughout the developing brain, blocking glutamate-mediated synaptic transmission involving NMDA receptors markedly increases normally occurring neuronal death (Ikonomidou et al., 1999; Heck et al., 2008). The mechanism involves a reduction of neuronal expression of anti-apoptotic proteins, such as B-cell lymphoma 2 (BCL-2; Hansen et al., 2004). Conversely, a limited increase in neuronal activity leads to down-regulation of the pro-apoptotic genes BAX and caspase 9 (Léveillé et al., 2010), thereby reducing the propensity of these cells to initiate programmed cell death (Hardingham et al., 2002). In addition to directly modulating the expression of apoptotic proteins, neuronal activity affects the expression of several secreted growth factors, such as BDNF (Hardingham et al., 2002; Hansen et al., 2004) and GDNF (Léveillé et al., 2010). So, even though BDNF is not a major survival factor in the developing CNS, it appears to be critical for activity-dependent neuroprotection (Tremblay et al., 1999). A recent publication revealed that certain populations of neurons in the CNS do not follow the predictions of the neurotrophic theory and showed that apoptosis of cortical inhibitory neurons is independent of cues present in the developing cerebral cortex (Southwell et al., 2012). This study indicates that programmed cell death of a large proportion of interneurons in the CNS is regulated by intrinsic mechanisms that are largely resistant to the presence or absence of extrinsic cues (Dekkers and Barde, 2013).Taken together, even though the extent of naturally occurring cell death in the different regions of the CNS is not nearly as well characterized as in the PNS, let alone quantified, it appears that its regulation may significantly differ. Although single secreted neurotrophic factors seem to be largely dispensable for survival, neuronal activity and other intrinsic mechanisms drive the propensity of the neurons in the CNS to undergo apoptosis. An important open question in this context is a possible involvement of non-neuronal cells, such as glial cells (see Corty and Freeman, in this issue).

The apoptotic machinery as a regulator of connectivity

Activation of the executor caspases has been most studied in cell bodies and typically results in the demise of the entire cell (Williams et al., 2006). However, recent evidence shows that caspases are also activated locally in neuronal processes and branches destined to be eliminated, for example in axons overshooting their targets that are subsequently pruned back to establish the precise adult connectivity (Finn et al., 2000; Raff et al., 2002; Luo and O’Leary, 2005; Buss et al., 2006b). Initially, axonal degeneration and axon pruning were thought to be independent of caspases (Finn et al., 2000; Raff et al., 2002). Later work in Drosophila melanogaster (Kuo et al., 2006; Williams et al., 2006) and in mammalian neurons (Plachta et al., 2007; Nikolaev et al., 2009; Vohra et al., 2010) demonstrated that interfering with the apoptotic balance or the executor caspases can prevent or at least delay axonal degeneration. Simon et al. (2012) have found that a caspase 9 to caspase 3 cascade is crucial for axonal degeneration induced by NGF withdrawal, with caspase 6 activation playing a significant but subsidiary role. Upstream of the caspases, BCL-2 family members such as BAX and BCL-X_l are required (Nikolaev et al., 2009; Vohra et al., 2010). It is conceivable that the failure of ipRGCs in BAX-deficient mice to form appropriate connections to other cells in the retina (Chen et al., 2013) may be in part attributable to defective axonal degeneration. Surprisingly, Apaf1 appears not to be involved in this process (Cusack et al., 2013), suggesting that axon degeneration depends on the concerted activation of the intrinsic initiator complex in a different way from apoptosis.Strikingly, a series of recent studies showed that several caspases and components of the intrinsic pathway also affect normal synaptic physiology in adulthood (Fig. 3, A–D). Here, pro-apoptotic proteins are predominantly involved in weakening the synapses, whereas the anti-apoptotic proteins have been mainly associated with synaptic strengthening (Fig. 3 B). In particular, caspase 3 promotes long-term depression (LTD), a stimulation paradigm that results in a period of decreased synaptic transmission (Li et al., 2010), and also prevents long-term potentiation (LTP), the converse situation leading to strengthened synaptic transmission (Jo et al., 2011). Likewise, the proapoptotic BCL-2 family members BAX and BAD stimulate LTD (Jiao and Li, 2011). By contrast, the anti-apoptotic protein BCL-X_l increases synapse numbers and strength (H. Li et al., 2008), and the inhibitor of apoptosis protein (IAP) family member survivin was reported to be involved in LTP in the hippocampus (Iscru et al., 2013) and in activity-dependent gene regulation (O’Riordan et al., 2008).Open in a separate windowFigure 3.Canonical and noncanonical functions of the apoptotic machinery. (A) The apoptotic machinery is not only involved in eliminating cells destined to die, but is also a central player in refining neuronal connectivity, by regulating synaptic transmission and by generating the adult connectivity through axon pruning (Luo and O’Leary, 2005; Hyman and Yuan, 2012). But how the canonical and noncanonical roles of the apoptotic machinery are interlinked and spatially restricted is not well understood. (B) In the adult nervous system, the pro-apoptotic proteins BAX, caspase 9, and caspase 3 promote weakening of synapses (long-term depression [LTD]; Li et al., 2010; Jiao and Li, 2011; Jo et al., 2011), while the anti-apoptotic proteins Bcl-X_l and the IAP survivin promote synaptic strengthening (long-term potentiation [LTP]; Li et al., 2008a; Iscru et al., 2013). It is unclear how the activation of these pathways is restricted to a single synapse, but a recent review suggested that the proteasomal degradation of activated caspases may prevent their diffusion (Hyman and Yuan, 2012). (C) Caspase activation is now known to be required for axon pruning during development to generate the adult refined connectivity (Luo and O’Leary, 2005; Simon et al., 2012). Different pathways are activated depending on the stimulus leading to degeneration. Growth factor deprivation during development leads to activation the executor caspases 3 and 6 (Simon et al., 2012) through the intrinsic apoptotic pathway, although its core protein Apaf1 does not seem to be required for this process (Cusack et al., 2013). On the other hand, a traumatic injury leads to reduced influx of NMNAT2 into the axon, which negatively affects the stability and function of mitochondria and leads to an increased calcium concentration (Wang et al., 2012). The effector caspase, caspase 6, is dispensable for this form of axonal degeneration (Vohra et al., 2010; Simon et al., 2012). Regulatory proteins such as the IAPs and also the proteasome seem to play a role in limiting the extent of activation to the degenerating part of the axon (Wang et al., 2012; Cusack et al., 2013; Unsain et al., 2013). (D) Simplified schematic of the main pro- and anti-apoptotic components. DISC, death-induced signaling complex. IAP, inhibitor of apoptosis protein. See Fig. 1 for details.These findings indicate that the apoptotic machinery acts at different levels in the cell, ranging from driving sub-lethal degradation of a compartment (Fig. 3 C) and attenuating synaptic transmission at the neuronal network level (Fig. 3 B) to destroying the entire cell during development or in disease (Fig. 3 D). How the cell spatially restricts the extent of activation of the apoptotic machinery is yet unclear. For example, elimination of the somata of developing neurons after neurotrophin deprivation is preceded by axonal degeneration, but not all instances of axonal degeneration lead to the death of the neuron (Campenot, 1977; Raff et al., 2002). Local regulation of caspase activation by IAPs is well established as a means for ensuring the elimination of neuronal processes in D. melanogaster (Kuo et al., 2006; Williams et al., 2006). Recent findings suggest a similar role for IAP in mammalian neurons, where it limits caspase activation to the degenerating axon (Fig. 3 C; Cusack et al., 2013; Unsain et al., 2013). The spontaneous mutation Wallerian degeneration slow (WldS; Lunn et al., 1989) has been instrumental to understand that trauma-induced axon degeneration is a regulated process different from, and independent of, cell body degeneration (Wang et al., 2012), but also distinct from axon pruning (Hoopfer et al., 2006). Work on the chimeric protein encoded by the WldS mutation also led to the identification of the protein NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2) as a labile axon survival factor (Gilley and Coleman, 2010). How the WldS chimeric protein and NMNAT2 result in axon protection is unclear, but several lines of evidence seem to converge on local regulation of mitochondrial function and motility (Avery et al., 2012; Fang et al., 2012).Related to the spatial limiting of apoptotic activity is the question of how a local source of neurotrophins leads to the rescue of a developing peripheral neuron. When neurons encounter a source of neurotrophins, only the receptors close to the target will be activated, whereas the others, located further away, are not. The cell, therefore, needs to integrate a pro-survival signal from the activated receptors, and death-inducing signals from the nonactivated dependence receptors. The continued signaling of activated neurotrophin receptors that are retrogradely transported to the soma (Grimes et al., 1996; Howe et al., 2001; Wu et al., 2001; Harrington et al., 2011) likely play a role in counteracting the pro-apoptotic signaling proximal to the source of neurotrophins. It will be interesting to investigate whether similar mechanisms play a role in axon pruning and traumatic axon degeneration as well.

Programmed cell death in the adult brain

Most of the nervous system becomes post-mitotic early in development. In rodents, two brain areas retain the capacity to generate new neurons in the adult: the sub-ventricular zone, which generates neurons that migrate toward the olfactory bulb, and the sub-granular zone of the dentate gyrus of the hippocampus, where neurons are generated that integrate locally. Similar to what is observed during embryonic development, these adult-generated neurons are produced in excess, and a large fraction undergoes apoptosis when contacting its designated targets (Petreanu and Alvarez-Buylla, 2002; Kempermann et al., 2003; Ninkovic et al., 2007). Preventing apoptosis of adult-generated neurons in the olfactory bulb only has limited functional consequences (Kim et al., 2007), whereas a similar maneuver in the dentate gyrus does lead to impaired performance in memory tasks (Kim et al., 2009). Why superfluous hippocampal neurons would need to be eliminated for proper function is a matter of speculation, but may be linked with the fact that these are excitatory projection neurons, whereas in the olfactory bulb only axon-less inhibitory granule cells are integrated. The extent of survival in both these areas critically depends on the activity of the neuronal network in which these newly born neurons have to integrate (Petreanu and Alvarez-Buylla, 2002; Kempermann et al., 2006; Ninkovic et al., 2007). In this context, BDNF, the expression level of which is well known to be regulated by network activity, supports the survival of young adult–generated neurons and possibly even stimulates the proliferation of neural progenitors (Y. Li et al., 2008; Waterhouse et al., 2012). Interestingly, in young adult mouse mutants that exhibit spontaneous epileptic seizures, significantly higher levels of BDNF have been measured (Lavebratt et al., 2006; Heyden et al., 2011). Concomitantly, the entire hippocampal formation is considerably enlarged by as much as 40% (Lavebratt et al., 2006; Angenstein et al., 2007), which in turn is dependent on the epileptic seizures (Lavebratt et al., 2006). Whether or not there is a causal relationship between increased BDNF levels and hippocampal volume remains to be established.

Conclusion

Now that is has become clear that action of the apoptotic machinery can be limited spatially and temporally, several questions need to be addressed: how do neurons integrate intrinsic and extrinsic pro- and anti-apoptotic signals; and how they are spatially restricted to allow degradation of a dendrite or axon, or modulation of synaptic transmission? Another important issue is the regulation of cell death by intrinsic mechanisms in the central nervous system of vertebrates, not least because programmed cell death is observed in the CNS in a number of neurodegenerative diseases (Vila and Przedborski, 2003). Indeed, several of the central apoptotic components discussed here are also involved in these disorders (Hyman and Yuan, 2012). New insights in the regulation of programmed cell death in the developing nervous system may therefore continue to help to better understand the pathophysiological mechanisms of neurodegenerative disorders.